How much nucleoside triphosphate is required to form one peptide bond during protein synthesis?

How much nucleoside triphosphate is required to form one peptide bond during protein synthesis?

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I'm trying to find out how many molecules of nucleoside triphosphates (ATP, GTP, UTP and/or CTP) it takes to release enough energy to link two amino acid monomers together with a peptide bond, specifically during the process of mRNA translation.

I've tried to do some research online, but I could not find a reputable source that will say definitely how much energy is consumed in the process. The best answer I could find is formulated based on 'Molecular Biology of the Cell' 4th edition by Alberts B, Johnson A, Lewis J, et al., which is that at least one molecule of ATP is consumed for every peptide linkage. Is this correct?

I've also read on a science forum that the amount of ATP consumed during translation is different for every amino acid, but I could not find a reliable source to back up that claim. Is this true?

Although the question shows considerable effort to achieve clarity, the way it is phrased as:

How many molecules of nucleoside triphosphate… [does] it take to release enough energy

still allows ambiguity, as I would not really regard the NTPs involved in protein synthesis “releasing energy”. So let us consider two reformulations of the question, as the explanation of the answers is of more scientific interest than the actual answers.

1. How many molecules of NTP are hydrolysed in the reactions causing the formation of one peptide bond on the ribosome?

Answer = 3

Formation of each peptide bond involves a cycle consisting of the introduction of a single new aminoacyl-tRNA to the A site of a ribosome carrying a growing polypeptide chain (or initiator tRNA for the first peptide bond), the peptidyl transferase reaction, and than translocation of the extended peptidyl-tRNA from A- to P-site. (See, e.g. Berg et al. online - Ch. 29)

1 ATP is hydrolysed in the aminoacylation reaction:

Amino Acid + tRNA + ATP → Aminoacyl-tRNA + AMP + PPi

1 GTP is hydrolysed in the aatRNA binding reaction catalysed by EF-Tu/EF1.

1 GTP is hydrolysed in the translocation reaction catalysed by EF-G/EF2

No NTP is consumed directly in the peptidyl transferase reaction - the energy for bond formation comes from the 'activated' aminoacyl-tRNA.

2. What is the total energetic cost in molecules of ATP for the formation of one peptide bond?

Here one might argue that:

Answer = 4+

The additional ATP occurs if one considers the total energetic cost of the aminoacylation reaction as 2 ATP, not 1 ATP. This arises from the fact that the ATP is hydrolysed to AMP (+PPi) and not ADP. Recycling of the AMP involves first the use of 1 molecule of ATP in the adenylate kinase reaction to produce ADP:


followed by the energy (from membrane ATP synthase) to regenerate ATP from ADP:

ADP + Pi ⇾ ATP

Why 4+? Certain amino acids (e.g. val and ile) are sufficiently similar to one another that the aminoacyl-tRNA synthetases have evolved a proof reading capacity, in which any incorrectly aminoacylated tRNA is hydrolysed. This only occurs for certain amino acids and at a rate that is difficult to determine, so the wastage of the ATP in this manner cannot be calculated precisely.

First, during the initiation of translation, a small ribosomal subunit binds to a molecule of mRNA. In a bacterial cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine. And then, this process need one molecular GTP, and GTP--->GDP+Pi, which can provide energy for the assembly. You can find more details in the book Campbell Biology, by Reece, Urry,…

About 5 ATP molecules are required for the addition of a single amino aid to a growing peptide chain.

I found this answer in Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis by Piques et al.:

The addition of an amino acid to a growing peptide chain requires two ATP molecules for amino acid activation and another two ATP for peptide bond formation and ribosome translation, plus additional costs of about another ATP, for error correction and the synthesis of sequences that are removed during protein maturation.

Actually it depends on the question. Whether you mean to say: How many amino acids are required? Or proteins? There is a characteristic difference between amino acids and proteins. Hence the number. Of amino acids can be determined by the following- If we consider a protein composed of "n" number of amino acids, it takes (4n)-1 number of ATP for the translation process.

4 ATP : 2 for activation amino acids to bind with specific tRNA. 1 for initiation 1 for elongation to push tRNA to the p site of ribosome

  • Adenosine triphosphate is composed of the nitrogenous base adenine, the five-carbon sugar ribose, and three phosphate groups.
  • ATP is hydrolyzed to ADP in the reaction ATP+H2O&rarrADP+Pi+ free energy the calculated ∆G for the hydrolysis of 1 mole of ATP is -57 kJ/mol.
  • ADP is combined with a phosphate to form ATP in the reaction ADP+Pi+free energy&rarrATP+H2O.
  • The energy released from the hydrolysis of ATP into ADP is used to perform cellular work, usually by coupling the exergonic reaction of ATP hydrolysis with endergonic reactions.
  • Sodium-potassium pumps use the energy derived from exergonic ATP hydrolysis to pump sodium and potassium ions across the cell membrane while phosphorylation drives the endergonic reaction.
  • energy coupling: Energy coupling occurs when the energy produced by one reaction or system is used to drive another reaction or system.
  • endergonic: Describing a reaction that absorbs (heat) energy from its environment.
  • exergonic: Describing a reaction that releases energy (heat) into its environment.
  • free energy: Gibbs free energy is a thermodynamic potential that measures the useful or process-initiating work obtainable from a thermodynamic system at a constant temperature and pressure (isothermal, isobaric).
  • hydrolysis: A chemical process of decomposition involving the splitting of a bond by the addition of water.

Chapter 3: Gene expression and protein synthesis

The last chapter pointed out that proteins are the cogs of all cellular machinery. Proteins are also the most diverse biomolecules with regard the their functionality. Just about every cellular process has a bunch of proteins helping it along. So where do these guys come from? Well, in case you slept through Introduction to Biology, the answer is that DNA encodes the sequence of amino acids that makes up every protein we know about. In other words, proteins are synthesized according to instructions stored as genes in a cell’s DNA.

There’s a long, multi-step process that takes us from a strand of DNA to a protein. The two key processes are transcription, where DNA encodes a strand of RNA and translation, where the aforementioned RNA directs the synthesis of a chain of amino acids (i.e., a protein). There are plenty of online resources that explain this process, but here it is in a nutshell (We’ll circle back and touch on some of these phenomena in a bit).

  1. A gene (double-stranded DNA) in the cell nucleus gets copied to a single-stranded RNA chain called messenger RNA (mRNA for short) 1 .
  2. The mRNA gets cut up and stitched together to remove certain sequences of non-coding nucleotides called introns and keep the actual coding sequences called exons.
  3. The mRNA leaves the nucleus and hooks up with the smaller of two ribosomal subunits 2 .
  4. The first amino acid (always methionine in eukaryotes) is ushered over to the mRNA ribosome complex by transfer RNA (or tRNA). There are special tRNA molecules for each type of amino acid, and each tRNA has a sequence of three nucleotides that matches up with each sequence of three nucleotides (called a codon) on the mRNA.
  5. The larger ribosomal subunit attaches to the smaller one, bringing with it binding sites for charged (amino-acid bearing) tRNAs 3 .
  6. A charged tRNA matches up with the next codon and gets bound to the ribosome using energy from a guanosine triphosphate (GTP – similar to an ATP molecule 4 .
  7. The two amino acids on the tRNAs are forced together such that a peptide bond forms between them.
  8. The first tRNA disassociates from its methionine molecule and the mRNA-ribosome complex as the mRNA slides over to reposition the remaining tRNA and the next codon into the appropriate binding sites 5 .
  9. The elongation phase proceeds as the next charged tRNA binds to the ribosome with the help of another GTP and an elongation factor, and a peptide bond forms between the next two amino acids 6 . This process repeats for as long as there are unprocessed codons on the mRNA.
  10. Termination of the process happens when the ribosome faces a stop codon (UAA, UAG, or UGA). Releasing factor recognizes this codon and causes the release of the polypeptide chain.
  11. The ribosome-mRNA complex then dissociates and the two ribosomal subunits can move on to another mRNA.

Below are a few good resources that can help you understand protein synthesis. As you go over these resources and/or any you find on your own, bear in mind that there are several important differences between protein synthesis in 7 cells. Among these differences are the types and number of initiation factors that help set up protein synthesis, the types of ribosomal subunits, and the location where the process takes place. So if you come across what seem to be conflicting explanations, make sure they are talking about the same kinds of cells.

Regulation of gene expression

So our cells can make proteins, but how do they know which proteins to make and how much to make of each one? Gene expression refers to the production of proteins from a given part of the genome, and regulation of gene expression is vital to maintaining that all important state of homeostasis. Just about every step in the pathway from DNA to mRNA to protein synthesis involves regulatory mechanisms that dictate when a gene is expressed and how much product it should yield.

A primary gene regulation mechanism involves promoter regions within the genome. For a gene to be transcribed to mRNA, a protein complex called RNA polymerase must bind at a promoter site near the sequence to be transcribed. Promoters contain specific DNA sequences that match up with particular RNA polymerases as well as proteins called transcription factors that sometimes help attract RNA polymerase to the right spot. There can also be transcription factors located far away from the actual gene that would be transcribed. These transcription factors have specific activator or repressor sequences of corresponding nucleotides, so they can effectively turn genes on and off and thus regulate gene expression.

Here’s a nice video that illustrates the interactions of a promoter, RNA polymerase, and a transcription factor.

Gene regulation also happens after mRNA is transcribed. Most mRNA has to have non-coding sequences (introns) removed before it leaves the nucleus for translation. The cutting and pasting involved in the process is mediated by spliceosomes, which consist of numerous small nuclear DNA (snDNA) subunits and proteins. In metazoans, there are major spliceosomes and minor spliceosomes. The major splicesosomes are common and do most of the work. But the minor spliceosome is rare, and usually doesn’t get much attention. However, some recent research indicates that the minor spliceosome appears to play a major regulatory role in a range of important gene products. The activity of the minor spliceosome can serve as a bottleneck to slow down or speed up protein synthesis. By manipulating levels of the snDNA subunit called U6atac, researchers 8 were able to control expression of transcription regulators, ion channels, signaling proteins, and DNA damage-repair proteins. They also found that expression of U6atac responds to cellular requirements for gene products. For instance, during periods of stress, U6atac is upregulated.

Regulation at the pre-translation stage is also affected by other methods. Before an mRNA strand leaves the nucleus, it has to get a cap on its 5′ end (only in eukaryotes), and it has to get a poly(A) tail attached to the other end. 9 Both of these processes can be held up or advanced as a means of regulating gene expression. Also, it is possible for cells to simply retain RNA transcripts in the nucleus so they cannot get to the cytoplasm where the ribosomes are.

Finally, gene regulation can occur at the translation phase, usually at the level of initiation. For example, proteins can bind to mRNA that inhibit or promote the formation of the mRNA-ribosome complex. The secondary structure of the mRNA 10 can determine how well it links up with binding proteins and/or ribosomal subunits, and this secondary structure can change in response to chemical conditions in the cell, such as temperature. Hence, regulation can be quick and immediate.

ATP and Energy Coupling

Exactly how much free energy (∆G) is released with the hydrolysis of ATP, and how is that free energy used to do cellular work? The calculated ∆G for the hydrolysis of one mole of ATP into ADP and Pi is &minus7.3 kcal/mole (&minus30.5 kJ/mol). However, this is only true under standard conditions, and the ∆G for the hydrolysis of one mole of ATP in a living cell is almost double the value at standard conditions: 14 kcal/mol (&minus57 kJ/mol).

ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP + Pi, and the free energy released during this process is lost as heat. To harness the energy within the bonds of ATP, cells use a strategy called energy coupling.

Cells couple the exergonic reaction of ATP hydrolysis with the endergonic reactions of cellular processes. For example, during cellular metabolic reactions, or the synthesis and breakdown of nutrients, certain molecules must be altered slightly in their conformation to become substrates for the next step in the reaction series. In the very first steps of cellular respiration, glucose is broken down through the process of glycolysis. ATP is required for the phosphorylation of glucose, creating a high-energy but unstable intermediate. This phosphorylation reaction causes a conformational change that allows enzymes to convert the phosphorylated glucose molecule to the phosphorylated sugar fructose. Fructose is a necessary intermediate for glycolysis to move forward. In this example, the exergonic reaction of ATP hydrolysis is coupled with the endergonic reaction of converting glucose for use in the metabolic pathway.


Energy transfer Edit

GTP is involved in energy transfer within the cell. For instance, a GTP molecule is generated by one of the enzymes in the citric acid cycle. This is tantamount to the generation of one molecule of ATP, since GTP is readily converted to ATP with nucleoside-diphosphate kinase (NDK). [1]

Genetic translation Edit

During the elongation stage of translation, GTP is used as an energy source for the binding of a new amino-bound tRNA to the A site of the ribosome. GTP is also used as an energy source for the translocation of the ribosome towards the 3' end of the mRNA. [2]

Microtubule dynamic instability Edit

During microtubule polymerization, each heterodimer formed by an alpha and a beta tubulin molecule carries two GTP molecules, and the GTP is hydrolyzed to GDP when the tubulin dimers are added to the plus end of the growing microtubule. Such GTP hydrolysis is not mandatory for microtubule formation, but it appears that only GDP-bound tubulin molecules are able to depolymerize. Thus, a GTP-bound tubulin serves as a cap at the tip of microtubule to protect from depolymerization and, once the GTP is hydrolyzed, the microtubule begins to depolymerize and shrink rapidly. [3]

Mitochondrial function Edit

The translocation of proteins into the mitochondrial matrix involves the interactions of both GTP and ATP. The importing of these proteins plays an important role in several pathways regulated within the mitochondria organelle, [4] such as converting oxaloacetate to phosphoenolpyruvate (PEP) in gluconeogenesis. [ citation needed ]

→ Gene is a macromolecule attached to an undifferentiated protein thread (chromonema) that can pass from one cell to another and can be transmitted from one generation to another without being changed at all.

→ Gene is a unit of heredity and responsible for inheritance.

→ Gene is a part of DNA and is made up of nucleotides. It can grow, reproduce and mutate. Gene is the unit of recombination. Genetic information is conveyed from DNA to mRNA.

→ Genes mainly act by producing proteins or enzymes which are required at various levels of metabolism. Proteins are made up of polypeptides which are formed from amino acids. Genes determine the physical and physiological characteristics of living beings.

→ DNA and RNA are two types of nucleic acids, which are polymers of nucleotides. DNA is the genetic material in most organisms. RNA is found only in some viruses as genetic material, it acts as a messenger to transfer the genetic information from DNA to proteins. It also functions as an adapter, structural and catalytic molecule.

→ DNA (deoxyribonucleic acid) is the polymer of deoxyribonucleotides. The haploid content of human DNA is 3.3 × 10 9 bp. DNA is chemically and structurally more stable than RNA.

→ Nucleic acids were first isolated by Friedrich Miescher (1869) from pus cells. These were named nuclein. Due to their acidic nature, they were named nucleic acid, and Altmann (1899) named them nucleic acids. Fisher (1880s) discovered the presence of purine and pyrimidine bases. Levene (1910) found deoxyribose nucleic acid contains phosphoric acid and deoxyribose sugar. He characterized four types of nucleotides.

Chargaff (1950) found that purines are equal to pyrimidines in DNA, and adenine = thymine, and guanine = cytosine. W.T. Astbury discovered through X-ray diffraction that DNA is a polynucleotide with nucleotides arranged perpendicular to the long axis of the molecule and are 0.34nm away from one another.

Wilkins and Franklin (1953) through X-ray photographs found that DNA was a helix with 2.0nm width. One turn of the helix was 3.4 nm with 10 layers of bases stacked in it. Through these photographs, Watson and Crick (1 953) built a three-dimensional model of DNA. They were awarded Nobel Prize in 1962. They purposed that DNA consists of a double helix with two chains having sugar-phosphate as the backbone and nitrogen bases on the inner sides.

The nitrogen bases of two chains form complementary pairs with purines of one and pyrimidine of the other held together by hydrogen bonds. The two chains are antiparallel with 5′ → 3′ orientation and 3′ → 5′ orientation. The two chains are twisted helically as a rope ladder with rigid steps twisted into a spiral. Each turn contains 10 nucleotides. In Vitro, DNA synthesis was carried out by Komberg (l 959). DNA has got an implicit mechanism for replication and copying.

→ A nucleotide is made up of three components: a nitrogenous base, a pentose sugar (ribose in RNA, deoxyribose in DNA), and a phosphate group. Nitrogenous bases are of two types: purines and pyrimidines. Purines are Adenine and Granite and pyrimidines are Cytosine, Uracil, and Thymine. Thymine is present in DNA. Thymine is replaced by Uracil in RNA.

→ A nucleoside is formed by a nitrogenous base linked to pentose sugar by an N-glycosidic bond, e.g. deoxyadenosine or adenosine, deoxycytidine, or cytidine.

Structure of DNA.

A. Chemical Structure and bonding of different constituents of DNA in the two chains.
B. sequence of nucleotides in a part of the double helix of DNA. C. coiling in double helix or duplex of DNA.

→ Two nucleotides are bound to each other through a phosphodiester bond. The phosphate group provides acidity to the nucleic acids. In Bacteria nucleotide, the DNA is covalently closed at its two ends. It’s called circular DNA. Its also present in mitochondria, plastids, and some viruses. Eukaryotic cells have free linear DNA. Both types of DNA are coiled and supercoiled to get into small space.

→ DNA replication is autocatalytic, it occurs during the S-phase of the cell cycle. Watson and Crick proposed a semiconservative model of replication for DNA. During which one strand of the daughter strand is derived from the parental duplex and the other strand is formed a new. Taylor et al (1957) worked with Broad Bean root tips, using

The experiment of Mcselson and Stahi (i958) to prove semi-conservative replication of DNA

Semi-conservative replication of DNA showing Zipper duplication

radiolabelled thymine and showed DNA replication is semi-conservative. In 1958 Nesselson and Stahl proved the semi-conservative mode of replication. They worked with E.Coli, using a heavy isotope of nitrogen, N 15 . E.Coli was grown with N 15 for several generations till the bacteria became completely labeled with N 15 . They were then shifted to medium with normal N 14 nitrogen.

The sample was taken after every generation and tested for the presence of N 15 and N 14 using density gradient centrifugation with CsCl. The first generation was found to be a hybrid between N1 15 and N 14 . The second-generation contained two types of DNA, 50% light (N 14 ) and 50% intermediate. This is only possible if two strands separate during replication and act as a template for the synthesis of new complementary strands. Thus proving a semiconservative mode of replication.

→ The process of replication starts at a particular spot known as the origin of replication. The deoxyribonucleotides are first phosphorylated and activated. Energy and phosphorylase enzymes are required for activation. Enzymes’ topoisomerases are specialized to break and reseal the DNA strands. Enzyme helicases unwind the DNA helix and separate the two strands. DNA binding proteins bind on the separated strands. The whole of DNA does not open up for replication, the point of separation proceeds from one end to another. The replication fork appears Y- Y-shaped during the process of replication.

Replication of DNA continuous over one strand and discontinuous over other strands

DNA polymerase enzyme requires an RNA primer to initiate the process/ RAN primer is synthesized at the 5′ end of new DNA strand by enzyme primase. The two separated strands act as templates. The new nucleotides are added according to the law of base pairing i.e. A pairs with T and G pairs with C.

Energy is utilized in forming hydrogen bonds between the free nucleotides and nitrogen bases of templates. Elongation of the DNA chain requires enzyme DNA polymerase III in the presence of Mg 2+ and ATP. The adjacent nucleotides attach to one another by phosphodiester bonds. As replication proceeds, the strands unwind and at the end, the RNA primer is removed and the gap is filled with the help of DNA polymerase 1. This is also called Zipper duplication.

DNA polymerase can act only in the 5′ → 3′ direction so replication is continuous on the strand (3′ → 5′) known as the leading strand. The other strand called lagging strand 1 (5′ → 3′) replicates itself in small stretches called Okazaki fragments. These fragments are joined by the DNA ligase enzyme. Any mismatch or mutation if occurs during replication can be corrected by proofreading and DNA repair mechanisms

→ Frederick Griffith (1928) by doing transforming experiments on mice with Streptococcus pneumonia found out the transfer of genetic material from a heat-killed infectious stain to the normal non-infectious stain.

→ Earlier the genetic material was thought to be protein. In 1933 – 44 Oswald Avery, Colin Macleod, and Maclyn McCarty discovered that the transforming biochemical in Griffith’s experiments was DNA.

→ Alfred Hershey and Martha Chase (1952) proved that DNA is the hereditary material. They worked with bacteriophage T, which infects E.Coli.

→ Francis Crick proposed the central dogma in molecular biology which explains the flow of genetic information
i.e. DNA → RNA → Protein.

→ The formation of RNA over a DNA template is called transcription. Transcription is meant for taking the coded information from DNA to the site where it is required for protein synthesis. Only one DNA strand transcribes RNA, it is called a sense strand. Transcription requires enzyme RNA polymerase. Prokaryotes have only one RNA polymerase. Eukaryotes have three RNA polymerases.

RNA polymerase 1 synthesizes rRNA, 28S, 18S, and 5.8S, RNA polymerase II synthesizes hnRNA, mRNA, and snRNAs, and RNA polymerase III synthesizes tRNA, 5SRNA, and siRNAs. A transcription unit consists of a promoter, the structural gene, and a terminator.

Schematic Structure of a Transcription Unit

→ In eukaryotes, the primary transcript contains exons and introns so it is non-functional. Exons are coding sequences whereas introns are intervening sequences. These introns are removed by splicing. It is followed by capping, methyl guanosine triphosphate addition to 5′ end and of hnRNA and tailing around 200 – 300 adenyl residues are added at 3′ end. The fully processed hnRNA is called mRNA and is transported out of the nucleus.

→ The relationship between the sequence of amino acids in a polypeptide and nucleotides sequence of DNA or mRNA is called genetic code.

→ George Gamow proposed that the genetic code is a triplet in nature. Marshall and Nirenberg’s method for cell-free protein synthesis helped to decipher the code. The chemical method developed by Har Gobind Khorana helped in synthesizing RNA molecules with defined base combinations. Severo Ochoa’s enzyme polynucleotide phosphorylase helped to polymerize RNA with defined sequences. All these methods helped in deciphering the genetic code.

→ The translation is the process of polymerization of amino acids to form a polypeptide. mRNA sequence decides the sequence of amino acids. Amino acids are joined through peptide bonds. Ribosomes are considered as the protein-synthesizing cellular factory. It requires amino acids, mRNA, tRNA, aminoacyl tRNA synthetase, and various enzymes along with ribosomes to complete the process of protein synthesis.

The process of translation occurs in the cytoplasm. The process takes place in several steps like activation of amino acids, initiation, elongation, and termination. Polyribosomes or polysomes help to produce a number of copies of the same polypeptide. In it, different ribosomes are held together by a strand of messenger RNA. These ribosomes usually form rosette or helical groups during active protein synthesis and are known as polyribosomes.

Ribosomes as a protein factory. A relationship between the various components. B. synthesis of the polypeptide on ribosome connected with the endoplasmic reticulum.

Cistron is a segment of DNA consisting of a stretch of base sequences that codes for one polypeptide chain, one transfer RNA (tRNA), ribosomal RNA tRNA) a molecule or performs any other specific function in connection with transcription, including controlling the functioning of other cistrons (operon model of gene action).

Regulation of gene expression may occur at various steps like

  1. transcriptional level,
  2. processing level (splicing),
  3. transport of mRNA from the nucleus to the cytoplasm.
  4. translational level.

An operon is a part of genetic material (or DNA) which acts a: a single regulated unit having one or more structural genes, an operator gene, a promoter gene, a regulator gene, a repressor, and an inducer or corepressor (from outside). Operons are of two types, inducible and repressive.

Lac operon is an example of an inducible operon system. In E.Col the enzyme beta-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, which is used as a source of energy. If the bacteria do not have lactose in the medium they won’t need beta-galactosidase enzyme. Thus the production of an enzyme (protein) is regulated by the presence of lactose (inducer).

→ Anticodon: A triplet of bases present on tRN A complementing with mRNA codon is called the anticodon.

→ A-site: It’s the site where the second and next amino-acyl-tRNA enters the ribosome.

→ Codon: Triplet of bases on mRNA, which codes for one amino acid.

→ Central dogma: Unidirectional flow of information from DNA to RNA to protein.

→ Cistron: A segment of DNA that determines a single polypeptide chain.

→ DNA polymerase: The enzyme playing a pivotal role in adding the building blocks to the primer in a sequence as guided by the DNA template. It can polymerize nucleotides only in the 5’ → ‘3’ direction.

→ Frameshift mutation: The mutations which are caused by shifting the entire reading frame by addition or deletion on the segment of DNA.

→ Genetic code: The genetic presentation of codon through which the information in RNA is decoded in a polypeptide chain.

→ Glycosylation: The addition of sugar residues by modification of certain proteins, which are released in the lumen and are trapped in Golgi vesicles.

→ Hydrogen bond: The bond between nitrogenous bases of DNA binding two nucleotide chains. It is a weak bond.

→ Inducer: An effector molecule responsible for the induction of enzyme synthesis at recognition sites, to prevent self-cleavage by a modified enzyme that recognizes the sites and methylates specific nucleotides at each site.

→ Jumping genes: These are genes that shuffle from one location to another.

→ Phosphodiester bond: The bond between two adjacent nucleotides of two adjacent sugar moieties at 3’ and 5’ positions with phosphoric acid.

→ Phenocopy: When a normal gene under a different set of environmental conditions copies down the phenotypic characters of a mutant.

→ Restriction enzyme: An endonuclease that recognizes specific nucleotide sequences in DNA and makes a double-strand cleavage of DNA molecule.

→ Regulatory gene: Any gene which regulates or modifies the activity of other genes.

→ Repression: The phenomenon in which the synthesis of a set of enzymes leading to a product is shut down if the product is present in plentiful amounts.

→ Rho-factor: The factor which is required for termination of RNA synthesis at some sites.

→ Structural gene: A gene that codes for a polypeptide.

→ Silent mutation: This kind of mutation does not cause any change in the protein.

→ Thalassemia: Haemoglobin-based genetic disorder which involves frameshift mutation in β-chain of hemoglobin.

→ Transformation: The process in which the cell takes up the segment of the naked DNA from its surroundings and incorporates it in its hereditary material and ultimately expresses the character specified by incoming DNA.

→ Wobble position: The position on the codon where mutation occurs at 3rd base of triplet which still permits the normal interaction with anticodon.

Adenosine triphosphate

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Adenosine triphosphate (ATP), energy-carrying molecule found in the cells of all living things. ATP captures chemical energy obtained from the breakdown of food molecules and releases it to fuel other cellular processes.

Cells require chemical energy for three general types of tasks: to drive metabolic reactions that would not occur automatically to transport needed substances across membranes and to do mechanical work, such as moving muscles. ATP is not a storage molecule for chemical energy that is the job of carbohydrates, such as glycogen, and fats. When energy is needed by the cell, it is converted from storage molecules into ATP. ATP then serves as a shuttle, delivering energy to places within the cell where energy-consuming activities are taking place.

ATP is a nucleotide that consists of three main structures: the nitrogenous base, adenine the sugar, ribose and a chain of three phosphate groups bound to ribose. The phosphate tail of ATP is the actual power source which the cell taps. Available energy is contained in the bonds between the phosphates and is released when they are broken, which occurs through the addition of a water molecule (a process called hydrolysis). Usually only the outer phosphate is removed from ATP to yield energy when this occurs ATP is converted to adenosine diphosphate (ADP), the form of the nucleotide having only two phosphates.

ATP is able to power cellular processes by transferring a phosphate group to another molecule (a process called phosphorylation). This transfer is carried out by special enzymes that couple the release of energy from ATP to cellular activities that require energy.

Although cells continuously break down ATP to obtain energy, ATP also is constantly being synthesized from ADP and phosphate through the processes of cellular respiration. Most of the ATP in cells is produced by the enzyme ATP synthase, which converts ADP and phosphate to ATP. ATP synthase is located in the membrane of cellular structures called mitochondria in plant cells, the enzyme also is found in chloroplasts. The central role of ATP in energy metabolism was discovered by Fritz Albert Lipmann and Herman Kalckar in 1941.

Peptide bonding of amino acids to form proteins and its origins


Peptide Bond Formation of amino acids in prebiotic conditions: another unsurmountable problem of protein synthesis on early earth

Claim: If enough people (planets) play the lottery . even if the odds are 1 in a trillion. not only is winning (life) probable, it's almost a certainty
Reply: The Origin of life enthusiasts by unguided means making that claim are not aware that the formation of just one protein depends on bonding one amino acid to another, and the scientific evidence has demonstrated that this is not possible prebiotically.

In life today, polymerization occurs in the aqueous cytoplasm of cells, with ribosomes synthesizing proteins and a variety of polymerases synthesizing nucleic acids. The linking bonds of these polymers are peptide and ester bonds. In both cases, the polymerization reaction is thermodynamically uphill, with hydrolysis being favored. How then can polymers be synthesized? The answer, of course, is that the monomers have been chemically activated by input of metabolic energy so that polymerization is spontaneous in the presence of the enzymes or ribosomes that catalyze polymerization. A plausible mechanism for the synthesis of peptide bonds and ester bonds on the prebiotic Earth continues to be a major gap in our understanding of the origin of life. 22

Cairns-Smith, the Genetic Takeover, page 59:
For one overall reaction, making one peptide bond, there about 90 distinct operations are required. If you were to consider in more detail a process such as the purification of an intermediate you would find many subsidiary operations — washings, pH changes, and so on.

1. The synthesis of proteins and nucleic acids from small molecule precursors, and the formation of amide bonds without the assistance of enzymes represents one of the most difficult challenges to the model of pre-vital ( chemical) evolution, and for theories of the origin of life.
2. The best one can hope for from such a scenario is a racemic polymer of proteinous and non-proteinous amino acids with no relevance to living systems.
3. Polymerization is a reaction in which water is a product. Thus it will only be favored in the absence of water. The presence of precursors in an ocean of water favors depolymerization of any molecules that might be formed.
4. Even if there were billions of simultaneous trials as the billions of building block molecules interacted in the oceans, or on the thousands of kilometers of shorelines that could provide catalytic surfaces or templates, even if, as is claimed, there was no oxygen in the prebiotic earth, then there would be no protection from UV light, which would destroy and disintegrate prebiotic organic compounds. Secondly, even if there would be a sequence, producing a functional folding protein, by itself, if not inserted in a functional way in the cell, it would absolutely no function. It would just lay around, and then soon disintegrate. Furthermore, in modern cells proteins are tagged and transported on molecular highways to their precise destination, where they are utilized. Obviously, all this was not extant on the early earth.
5. To form a chain, it is necessary to react bifunctional monomers, that is, molecules with two functional groups so they combine with two others. If a unifunctional monomer (with only one functional group) reacts with the end of the chain, the chain can grow no further at this end. If only a small fraction of unifunctional molecules were present, long polymers could not form. But all ‘prebiotic simulation’ experiments produce at least three times more unifunctional molecules than bifunctional molecules.

The polymerization problem
Given an ocean full of small molecules of the types likely to be produced on pre-biological earth with the types of processes postulated by the origin of life enthusiasts, we must next approach the question of polymerization. This question poses a two-edged sword: we must first demonstrate that macromolecule synthesis is possible under pre-biological conditions, then we must construct a rationale for generating macromolecules rich in the information necessary for usefulness in a developing precell. We shall deal with these separately.1

The synthesis of proteins and nucleic acids from small molecule precursors represents one of the most difficult challenges to the model of pre-biological ( chemical) evolution.

There are many different problems confronted by any proposal. Polymerization is a reaction in which water is a product. Thus it will only be favored in the absence of water. The presence of precursors in an ocean of water favours depolymerization of any molecules that might be formed. Careful experiments done in an aqueous solution with very high concentrations of amino acids demonstrate the impossibility of significant polymerization in this environment.

Peptides by Activation of Amino Acids with CO on (Ni,Fe)S Surfaces: Implications for the Origin of Life
Claudia Huber and Guenter Waechtershaeuser
Under the dilute aqueous conditions most relevant for the origin of life, activation of the amino acids by coupling with hydrolysis reactions notably of inorganic polyphosphates has been suggested. It is, however, not clear how under
hot aqueous conditions such hydrolytically sensitive coupling compounds, if geochemically available at all, could resist rapid equilibration.

Abiotic origin of monomers – but what’s needed are polymers
Yet the formation of polymers in itself (regardless of the possible usefulness of their sequence) presents several problems which for the sake of clarity I shall discuss first in the context of amino acids and proteins, and comment later on the applicability of these comments to nucleotides and nucleic acids.

The most basic problem is that the amino acids must be able to join together, by linking the carboxyl group of one amino acid to the amino group of the next to form what is called a peptide bond (Figure 1). But this is a condensation reaction (involving loss of water) so it will not occur readily in an aqueous environment (such as a primeval soup) and it is significantly endothermic (energetically unfavourable), so it will not occur at all without the input of energy. This is why in the cell (a) ribosomes limit access of water to the active site where peptide bonds are formed [1], and (b) peptide bond formation is linked to the breaking of high-energy phosphate bonds so that the energy released in the latter can be used to enable the former. [2]
peptide bond

Figure 1. Formation of a peptide bond.

But forming the peptide bonds is only half of the problem. Because any scenario to try to generate biologically active proteins would require a plentiful supply of amino acids (not the meagre yield found in soup experiments) and some means of trying out different amino acid sequences (to try to find one with biological activity). That is, there needs to be means for breaking peptide bonds, to separate the amino acids, and then recombining them in a different sequence.
Abiotically, it requires many hours in hot mineral acid to hydrolyse peptide bonds (which is why proteins are so stable, and suitable for building biological tissues), but biologically this reaction is achieved readily with appropriate enzymes (e.g. the digestive enzyme trypsin).

So the point I am making here is that the conditions required to make and break peptide bonds are very different. That is, prebiotic scenarios would require transfer of the nascent polypeptides from one sort of environment to a chemically very different one, or some means of radically changing the conditions in the same environment (but without flushing out the polypeptides).

Whilst it is not too difficult to envisage possible situations (e.g. using ocean vents) that might have given the required different or changing conditions, at the very least this means that the volume where such ‘experiments’ might have taken place would have been severely limited – we certainly cannot envisage oceans of productive primeval soup.

As indicated here, a simple calculation shows that even with a virtually unlimited supply of amino acids and enzymatic production of proteins, the odds of producing a biologically active protein are practically hopeless. So how much more hopeless is the situation where prebiotic conditions are taken into account?

The concentration problem
Polymer formation in aqueous environments would most likely have been necessary on early Earth because the liquid ocean would have been the reservoir of amino acid precursors needed for protein synthesis. 1

A thermodynamic analysis of a mixture of protein and amino acids in an ocean containing a 1 molar solution of each amino acid (100,000,000 times higher concentration than we inferred to be present in the pre-biological ocean) indicates the concentration of a protein containing just 100 peptide bonds (101 amino acids) at equilibrium would be 10-338 molar. Just to make this number meaningful, our universe may have a volume somewhere in the neighbourhood of 10^85 litres. At 10-338 molar, we would need an ocean with a volume equal to 10229 universes (100, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000) just to find a single molecule of any protein with 100 peptide bonds. So we must look elsewhere for a mechanism to produce polymers. It will not happen in the ocean.

Sidney Fox, an amino acid chemist, and one of my professors in graduate school recognized the problem and set about constructing an alternative. Since water is unfavourable to peptide bond formation, the absence of water must favour the reaction. Fox attempted to melt pure crystalline amino acids in order to promote peptide bond formation by driving off water from the mix. He discovered to his dismay that most amino acids broke down to a tarry degradation product long before they melted. After many tries, he discovered two of the 20 amino acids, aspartic and glutamic acid, would melt to a liquid at about 200oC. He further discovered that if he were to dissolve the other amino acids in the molten aspartic and glutamic acids, he could produce a melt containing up to 50% of the remaining 18 amino acids. It was no surprise then that the amber liquid, after cooking for a few hours, contained polymers of amino acids with some of the properties of proteins. He subsequently named the product proteinoids. The polymerized material can be poured into an aqueous solution, resulting in the formation of spherules of protein-like material which Fox has likened to cells. Fox has claimed nearly every conceivable property for his product, including that he had bridged the macromolecule to cell transition. He even went so far as to demonstrate a piece of lava rock could substitute for the test tube in proteinoid synthesis and claimed the process took place on the primitive earth on the flanks of volcanoes. However, his critics, as well as his own students, have stripped his credibility. Note the following problems:

1) Proteinoids are not proteins they contain many non-peptide bonds and unnatural cross-linkages.
2) The peptide bonds they do contain are beta bonds, whereas all biological peptide bonds are alpha.
3) His starting materials are purified amino acids bearing no resemblance to the materials available in the "dilute soup." If one were to try the experiment with condensed "pre-biological soup," tar would be the only product.
4) The ratio of 50% Glu and Asp necessary for success in these experiments bears no resemblance to the vastly higher ratio of Gly and Ala found in nearly all primitive earth synthesis experiments.
5) There is no evidence of information contained in the molecules.

All of his claims have failed the tests of rationality when examined carefully. As promising as his approach seemed in theory, the reality is catastrophic to the hopes of paleo-biogeochemists.

A number of other approaches have been tried. The most optimistic of these is the use of clays. Clays are very thin, very highly ordered arrays of complex aluminium silicates with numerous other cations. In this environment, the basic amino groups tend to order and polymers of several dozen amino acids have been produced. While these studies have generated enthusiastic interest on the part of pre-biological evolutionists, their relevance is quickly dampened by several factors.

1) While ordered amino acids joined by peptide bonds result, the product contains no meaningful information.
2) The clays exhibit a preference for basic amino acids.
3) No polymerization of amino acids results if free amino acids are used.
4) Pure activated amino acids attached to adenine must be used in order to drive the reaction toward polymerization. Adenylated amino acids are not exactly the most likely substrate to be floating about the pre-biological ocean.
5) The resultant polymers are three dimensional rather than linear, as is required for biopolymers.

At least one optimistic scientist (Cairns-Smith, 1982) believes that the clay particles themselves formed the substance of the first organisms! In reality, the best one can hope for from such a scenario is a racemic polymer of proteinous and non-proteinous amino acids with no relevance to living systems.

The problem of chain termination
To form a chain, it is necessary to react bifunctional monomers, that is, molecules with two functional groups so they combine with two others. If a unifunctional monomer (with only one functional group) reacts with the end of the chain, the chain can grow no further at this end. If only a small fraction of unifunctional molecules were present, long polymers could not form. But all ‘prebiotic simulation’ experiments produce at least three times more unifunctional molecules than bifunctional molecules. Formic acid (HCOOH) is by far the commonest organic product of Miller-type simulations. Indeed, if it weren’t for evolutionary bias, the abstracts of the experimental reports would probably state nothing more than: ‘An inefficient method for production of formic acid is here described …’ Formic acid has little biological significance except that it is a major component of ant (Latin formica) stings.

A realistic prebiotic polymerisation simulation experiment should begin with the organic compounds produced by Miller-type experiments, but the reported ones always exclude unifunctional contaminants.

Dr Dudley Eirich comments:
I work in Biotech producing a bifunctional monomer for the polymer industry. I can attest to the fact that the final purified material for sale has to be essentially free of the monofunctional monomer. The final product generally has to be greater than 99.5% pure and for some applications the final product has to be greater than 99.9% pure. We have to use a lot of scientific knowledge and expensive equipment to attain those purity levels. Realistic ‘natural’ polymerization reactions could never produce long chains of polymers because there would always be overly high concentrations of monofunctional monomer components around to terminate growing chains.


Peptide bonding of amino acids to form proteins and its origins

Mystery of Life's Origin 4
Experimental evidence indicates that if there are bonding preferences between amino acids , they are not the ones found in natural organisms. There are three basic requirements for a biologically functional protein.
- One: It must have a specific sequence of amino acids . At best prebiotic experiments have produced only random polymers. And many of the amino acids included are not found in living organisms.
- Second: An amino acid with a given chemical formula may in its structure be either “righthanded” (D- amino acids ) or “left-handed” (L- amino acids ). Living organisms incorporate only L- amino acids . However, in prebiotic experiments where amino acids are formed approximately equal numbers of D- and L- amino acids are found. This is an “intractable problem” for chemical evolution (p. vi).
- Third: In some amino acids there are more positions than one on the molecule where the amino and carboxyl groups may join to form a peptide bond. In natural proteins only alpha-peptide bonds (designating the location of the bond) are found. In proteinoids, however, beta, gamma and epsilon peptide bonds largely predominate. Just the opposite of what one would expect if bonding preferences played a role in prebiotic evolution.

There is a huge gap that has to be filled between " modern " polypeptide formation through ribosomes, mRNA, and tRNA's, and supposed primordial amino chain formations without this advanced machinery. How could the gap be closed? Not only are prebiotic mechanisms unlikely, but the transition would require the emergence of all the complex machinery and afterward transition from one mechanism to the other. Tamura admits that fact clearly: the ultimate route to the ribosome remains unclear. It takes a big leap of faith to believe, that could be possible in any circumstances.

Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains
Proteins are linear polymers formed by linking the alpha-carboxyl group of one amino acid to the a-amino group of another amino acid. This type of linkage is called alpha peptide bond or an amide bond. The formation of a dipeptide from two amino acids is accompanied by the loss of a water molecule. The equilibrium of this reaction lies on the side of hydrolysis rather than synthesis under most conditions. Hence, the biosynthesis of peptide bonds requires an input of free energy. Nonetheless, peptide bonds are quite stable kinetically because the rate of hydrolysis is extremely slow the lifetime of a peptide bond in aqueous solution in the absence of a catalyst approaches 1000 years.

A series of amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit in a polypeptide is called a residue. A polypeptide chain has directionality because its ends are different: an alpha -amino group is present at one end and an a -carboxyl group at the other. The amino end is taken to be the beginning of a polypeptide chain by convention, the sequence of amino acids in a polypeptide chain is written starting with the amino-terminal residue. 12

Question: By the convention of whom .

Thus, in the polypeptide Tyr-Gly-Gly-Phe- Leu (YGGFL), tyrosine is the amino-terminal (N-terminal) residue and leucine is the carboxyl-terminal (C-terminal) residue . Leu- Phe-Gly-Gly-Tyr (LFGGY) is a different polypeptide, with different chemical properties.

Amino acid sequences have direction.
This illustration of the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL) shows the sequence from the amino terminus to the carboxyl terminus. This pentapeptide, Leu-enkephalin, is an opioid peptide that modulates the perception of pain. The reverse pentapeptide, Leu-Phe-Gly-Gly-Tyr (LFGGY), is a different molecule and has no such effects.

A polypeptide chain consists of a regularly repeating part, called the main chain or backbone, and a variable part, comprising the distinctive side chains (Figure below).

Components of a polypeptide chain.
A polypeptide chain consists of a constant backbone (shown in black) and variable side chains (shown in green).

The polypeptide backbone is rich in hydrogen-bonding potential. Each residue contains a carbonyl group (C = O), which is a good hydrogen-bond acceptor, and, with the exception of proline, an NH group, which is a good hydrogen-bond donor. These groups interact with each other and with functional groups from side chains to stabilize particular structures

Peptidyl transferase catalyzes peptide-bond synthesis
A molecule called the Peptidyl Transferase Center (PTC) is considered by some as having an essential role in the emergence of life, since this catalytic ability to get together amino acids is crucial for protein synthesis and thus, for the first transition from an RNA world to a Ribonucleoprotein world, as seen in modern organisms.

All known cellular organisms have the PTC conserved and the process of reading the information contained in the messenger RNA, in general, is similar in all life forms. Would the common ancestor of all life forms be a part of the largest subunit of the ribosomal RNA? When thinking about LUCA as a molecule, and more specifically, as the large subunit of the ribosome or even more specifically as the PTC, there is an extensive modification into the junction point on which all living organisms came to be. Here the nature of LUCA is changed since it places the common point of origin in a time where the RNA was the information-carrying molecule and the cellular systems were still starting to maturate. 10

The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate. 11

Positioning, ordering, providing, stabilizing, promoting a concerted shuttle mechanism are all tasks which we can easily attribute to the action of an intelligence, but could hardly emerge without external direction by random unguided events.

Protein synthesis in the cell is performed on ribosomes, large ribonucleoprotein particles that consist of three RNA molecules and more than 50 proteins. Ribosomes are composed of two subunits, the larger of which has a sedimentation coefficient of 50S in prokaryotes (the 50S subunit) and the smaller which sediments at 30S (the 30S subunit) together they form 70S ribosomes. The ribosome is a molecular machine that selects its substrates, aminoacyl-tRNAs (aa-tRNAs) d , rapidly and accurately and catalyzes the synthesis of peptides from amino acids. The 30S subunit contains the decoding site, where base-pairing interactions between the mRNA codon and the tRNA anticodon determines the selection of the cognate aa-tRNA.

The large ribosomal subunit contains the site of catalysis—the peptidyl transferase (PT) center—which is responsible for making peptide bonds during protein elongation and for the hydrolysis of peptidyl-tRNA (pepttRNA) during the termination of protein synthesis. The ribosome has three tRNA binding sites: A, P, and E sites ( figure below )

Schematic of Peptide Bond Formation on the Ribosome
The a-amino group of aminoacyl-tRNA in the A site (red) attacks the carbonyl carbon of the pept-tRNA in the P site (blue) to produce a new, one amino acid longer pept -tRNA in the A site and a deacylated tRNA in the P site. The 50S subunit, where the PT center is located, is shown in light gray and the 30S subunit in dark gray. A, P, and E sites of the ribosome are indicated.

During the elongation cycle of protein synthesis, aa-tRNA is delivered to the A site of the ribosome in a ternary complex e with elongation factor Tu (EF-Tu) c and GTP. Following GTP hydrolysis and release from EF-Tu, aa-tRNA accommodates in the A site of the Peptidyl Transferase Center ( PT center ) and reacts with pept-tRNA bound to the P site, yielding deacylated tRNA in the P site and A site pept-tRNA that is extended by one amino acid residue. The subsequent movement of tRNAs and mRNA through the ribosome (translocation) is catalyzed by another elongation factor (EF-G in bacteria). During translocation, pept-tRNA and deacylated tRNA move to the P and E sites, respectively a new codon is exposed in the A site for the interaction with the next aa-tRNA, and the deacylated tRNA is released from the E site.

The movement of aa-tRNA into the A site is a multistep process that requires structural rearrangements of the ribosome, EF-Tu, and aa-tRNA.

Structure of the Active Site of the Peptidyl Transferase Center (PTC)
50S subunits are composed of two rRNA molecules, 23S rRNA and 5S rRNA, and more than 30 proteins (Figure A below).

Structure of the Peptidyl Transferase Center
(A) Crystal structure of the 50S subunit from H. marismortui with a transition state analog (red) bound to the active site. Ribosomal proteins are blue, the 23S rRNA backbone is brown, the 5S rRNA backbone is olive, and
rRNA bases are pale green.
(B) Substrate binding to the active site. Base pairs formed between cytosine residues of the tRNA analogs in the A site (yellow) and P site (orange) with 23S rRNA bases (pale green) are indicated. The a-amino group of the A site substrate (blue) is positioned for the attack on the carbonyl carbon of the ester linking the peptide moiety of the P site substrate (green). Inner shell nucleotides are omitted for clarity.

The Mechanism of Peptide Bond Formation
The combined evidence supports the idea that peptide bond formation on the ribosome is driven by a favorable entropy change. The A and P site substrates are precisely aligned in the active center by interactions of their CCA sequences and of the nucleophilic a-amino group with residues of 23S rRNA in the active site. The most favorable catalytic pathway involves a six-membered transition state (Figure below) in which proton shuttling occurs via the 20-OH of A76 of the P site tRNA. The reaction does not involve chemical catalysis by ribosomal groups but may be modulated by conformational changes at the active site which can be induced by protonation.

Concerted Proton Shuttle Mechanism of Peptide Bond Formation
Pept-tRNA (P site) and aminoacyl-tRNA (A site) are blue and red, respectively, ribosome residues are pale green, and ordered water molecules are gray. The attack of the a-NH2 group on the ester carbonyl carbon results in a six-membered transition state in which the 20-OH group of the A site A76 ribose moiety donates its proton to the adjacent leaving 30 oxygen and simultaneously receives a proton from the amino group. Ribosomal residues are not involved in chemical catalysis but are part of the H bond network that stabilizes the transition state.

In addition to placing the reactive groups into close proximity and precise orientation relative to each other, the ribosome appears to work by providing an electrostatic environment that reduces the free energy of forming the highly polar transition state, shielding the reaction against bulk water, helping the proton shuttle forming the leaving group or a combination of these effects. With this preorganized network, the ribosome avoids the extensive solvent reorganization that is inevitable in the corresponding reaction in solution, resulting in significantly more favorable entropy of activation of the reaction on the ribosome.

With both the P site and the A site occupied by aminoacyl-tRNA, the stage is set for the formation of a peptide bond: the formylmethionine molecule linked to the initiator tRNA will be transferred to the amino group of the amino acid in the A site. The formation of the peptide bond, one of the most important reactions in life, is a thermodynamically spontaneous reaction catalyzed by a site on the 23S rRNA of the 50S subunit called the peptidyl transferase center. This catalytic center is located deep in the 50S subunit near the tunnel that allows the nascent peptide to leave the ribosome. The ribosome, which enhances the rate of peptide bond synthesis by a factor of 10^7 over the uncatalyzed reaction, derives much of its catalytic power from catalysis by proximity and orientation. The ribosome positions and orients the two substrates so that they are situated to take advantage of the inherent reactivity of an amine group (on the aminoacyl-tRNA in the A site) with an ester (on the initiator tRNA in the P site). The amino group of the aminoacyl-tRNA in the A site, in its unprotonated state, makes a nucleophilic attack on the ester linkage between the initiator tRNA and the formylmethionine molecule in the P site (Figure A below).

Peptide-bond formation.
(A) The amino group of the aminoacyl-tRNA attacks the carbonyl group of the ester linkage of the peptidyl-tRNA.
(B) An eight-membered transition state is formed. Note: Not all atoms are shown and some bond lengths are exaggerated for clarity.
(C) This transition state collapses to form the peptide bond and release the deacylated tRNA.

The nature of the transition state that follows the attack is not established and several models are plausible. One model proposes roles for the 2' OH of the adenosine of the tRNA in the P site and a molecule of water at the peptidyl transferase center (Figure B above). The nucleophilic attack of the a-amino group generates an eight-membered transition state in which three protons are shuttled about in a concerted manner. The proton of the attacking amino group hydrogen bonds to the 2' oxygen of ribose of the tRNA. The hydrogen of 2' OH, in turn, interacts with the oxygen of the water molecule at the center, which then donates a proton to the carbonyl oxygen. A collapse of the transition state with the formation of the peptide bond allows protonation of the 3'OH of the now empty tRNA in the P site (Figure C above). The stage is now set for translocation and formation of the next peptide bond.

The formation of a peptide bond is followed by the GTP-driven a translocation of tRNAs and mRNA
With the formation of the peptide bond, the peptide chain is now attached to the tRNA whose anticodon is in the A site on the 30S subunit. The two subunits rotate with respect to one another, and this structural change places the CCA b end of the same tRNA and its peptide in the P site of the large subunit (Figure below).

Mechanism of protein synthesis.
The cycle begins with peptidyltRNA in the P site.
(1) An aminoacyl-tRNA binds in the A site.
(2) With both sites occupied, a new peptide bond is formed.
(3) The tRNAs and the mRNA are translocated through the action of elongation factor G, which moves the deacylated tRNA to the E site.
(4) Once there, the tRNA is free to dissociate to complete the cycle.

Another aminoacyl-tRNA arrives and binds at the A site (1). Again, peptide bond synthesis occurs (2). However, protein synthesis cannot continue without the translocation of the mRNA and the tRNAs within the ribosome. Elongation factor G (EF-G, also called translocase) c catalyzes the movement of mRNA, at the expense of GTP hydrolysis, by a distance of three nucleotides. Now, the next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA (3). The peptidyl- tRNA moves out of the A site into the P site on the 30S subunit and at the same time, the deacylated tRNA moves out of the P site into the E site and is subsequently released from the ribosome (4). The movement of the peptidyl-tRNA into the P site shifts the mRNA by one codon, exposing the next codon to be translated in the A site.

The three-dimensional structure of the ribosome undergoes significant change during translocation, and evidence suggests that translocation may result from properties of the ribosome itself. However, EF-G accelerates the process. A possible mechanism for accelerating the translocation process is shown in Figure below.

Translocation mechanism.
In the GTP form, EF-G binds to the A site on the 50S subunit. This binding stimulates GTP hydrolysis, inducing a conformational change in EF-G that forces the tRNAs and mRNA to move through the ribosome by a distancecorresponding to one codon.

Question: How did unguided random processes select and finely tune the forces to move the tRNAs and mRNA by the right distance of one codon?

EF-G in the GTP form binds to the ribosome near the A site, interacting with the 23S rRNA of the 50S subunit. The binding of EF-G to the ribosome stimulates the GTPase activity of EF-G. On GTP hydrolysis, EF-G undergoes a conformational change that displaces the peptidyl-tRNA in the A site to the P site, which carries the mRNA and the deacylated tRNA with it. The dissociation of EF-G leaves the ribosome ready to accept the next aminoacyl-tRNA into the A site. Note that the peptide chain remains in the P site on the 50S subunit throughout this cycle, growing into the exit tunnel. This cycle is repeated, with mRNA translation taking place in the 5' ==>> 3' direction, as new aminoacyl-tRNAs move into the A site, allowing the polypeptide to be elongated until a stop signal is found.

The direction of translation has important consequences. Transcription also is in the 5' ==>> 3' direction. If the direction of translation were opposite that of transcription, only fully synthesized mRNA could be translated. In contrast, because the directions are the same, mRNA can be translated while it is being synthesized.

Question: How could natural, unguided, random processes select the right direction to be translated? Trial and error ?

In bacteria, almost no time is lost between transcription and translation. The 5' end of mRNA interacts with ribosomes very soon after it is made, well before the 3' end of the mRNA molecule is finished. An important feature of bacterial gene expression is that translation and transcription are closely coupled in space and time.

There is a huge gap that has to be filled between " modern " polypeptide formation through ribosomes, mRNA, and tRNA's, and supposed primordial amino chain formations without this advanced machinery. How could the gap be closed? Not only are prebiotic mechanisms unlikely, but the transition would require the emergence of all the complex machinery and afterward transition from one mechanism to the other. Tamura admits that fact clearly: the ultimate route to the ribosome remains unclear. It takes a big leap of faith to believe, that could be possible in any circumstances.

Mystery of Life's Origin 4
Experimental evidence indicates that if there are bonding preferences between amino acids , they are not the ones found in natural organisms. There are three basic requirements for a biologically functional protein.

One: It must have a specific sequence of amino acids . At best prebiotic experiments have produced only random polymers. And many of the amino acids included are not found in living organisms.

Second: An amino acid with a given chemical formula may in its structure be either “righthanded” (D- amino acids ) or “left-handed” (L- amino acids ). Living organisms incorporate only L- amino acids . However, in prebiotic experiments where amino acids are formed approximately equal numbers of D- and L- amino acids are found. This is an “intractable problem” for chemical evolution (p. vi).

Third: In some amino acids there are more positions than one on the molecule where the amino and carboxyl groups may join to form a peptide bond. In natural proteins only alpha-peptide bonds (designating the location of the bond) are found. In proteinoids, however, beta, gamma and epsilon peptide bonds largely predominate. Just the opposite of what one would expect if bonding preferences played a role in prebiotic evolution.

Studies of peptide bond formation in the absence of modern biological machinery can give insight into the mechanism employed by the ribosome’s active site, as well as yield important information in the prebiotic route to the first peptides in the origin of life. The formation of a peptide bond (reaction R1 shown below) is a condensation reaction, eliminating a water molecule for each peptide bond formed, and thus faces both thermodynamic and kinetic constraints in bulk aqueous solution

Amino Acids joined together through a dehydration reaction, where a water molecule is formed and removed to form a covalent bond called a peptide bond. A structure resulting from a bunch of these bonds repeating over and over is called a polypeptide. Like DNA molecules, polypeptides have a direction: they’ve got an amino acid at one end (the N-terminus) and a carboxyl group at the other (the C-terminus).

In modern biology, the condensation reactions necessary in the formation of peptide bonds are facilitated catalytically by the large subunit of the ribosome.

Fazale Rana's Cell's design: The chemical reactions that form the bonds that join amino acids together in polypeptide chains are catalyzed or assisted by ribosomes. The ribosome, mRNA, and tRNA molecules work cooperatively to produce proteins. Using an assembly-line process, protein manufacturing machinery forms the polypeptide chains (that constitute proteins) one amino acid at a time. This protein synthetic apparatus joins together three to five amino acids per second. Ribosomes, in conjunction with mRNA and tRNAs, assemble the cell's smallest proteins, about one hundred to two hundred amino acids in length, in less than one minute. The processing of proteins in the lumen (posttranslational modification) is quite extensive. Posttranslational modifications include (1) formation and reshuffling of disulfide bonds (these bonds form between the side chains of cysteine amino acid residues within a protein, stabilizing its three-dimensional structure)

Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain
Each amino acid is first coupled to specific tRNA molecules, next is the mechanism that joins these amino acids together to form proteins. The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Consequently, a protein is synthesized stepwise from its N-terminal end to its C-terminal end. Throughout the entire process, the growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (forming a peptidyl-tRNA). Each addition disrupts this high-energy covalent linkage, but immediately replaces it
with an identical linkage on the most recently added amino acid

The incorporation of an amino acid into a protein. A polypeptide chain grows by the stepwise addition of amino acids to its C-terminal end. The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the covalent attachment of a tRNA molecule. The peptidyl-tRNA linkage that activates the growing end is regenerated during each addition. The amino acid side chains have been abbreviated as R1, R2, R3, and R4 as a referencepoint, all of the atoms in the second amino acid in the polypeptide chain are shaded gray. The figure shows the addition of the fourth amino acid (red) to the growing chain.

Peptide Bond Formation: RNA's Big Bang

The genetic code may have been established gradually (Wong, 1975). 5

observe the " may have's ", by some means, might have, proposed the idea, would have,

The second law of thermodynamics indicates that peptide bond formation does not occur spontaneously. Therefore, energy must be added into the system by some means and amino acids must be "activated." Modern biological systems use the energy of the ATP hydrolysis for coupling many reactions (Lipmann, 1941). However, during the prebiotic stage, the light from the sun, geothermal energy, pressure in the thermal vent, or other similar sources may have been used in the process of activating the molecules of a system. The development of prebiotic precursors of biomolecules might have occurred in interstellar space, and were subsequently transferred to Earth by comets, asteroids, or meteorites (Oró, 1961 Chyba et al., 1990 Chyba & Sagan, 1992). Reactions on clay (Paecht-Horowitz et al., 1970) and/or dry mixtures of amino acids (Fox & Harada, 1958) may have facilitated the condensation of activated amino acids, thereby forming peptide bonds. Iron sulfate is known to cause unusual reducing reactions, especially with H2S. Wächtershäuser (1992) previously proposed the idea of an "iron-sulfur world" where low-molecular weight constituents may have originated autotrophic metabolism. In such circumstances, amino acids would have been converted into simple peptides (Huber & Wächtershäuser, 1998). In fact, it has been demonstrated that the peptide containing a thioester at the carboxyl-terminal undergoes nucleophilic attack by the side chain of the Cys residue at the amino terminal of another peptide. Moreover, the formed thioester ligation product readily undergoes a rapid intramolecular reaction at the α-amino group of the Cys to yield a product with a native peptide bond. This series of events is called "native chemical ligation" and is important in the general application of protein chemistry (Dawson et al., 1994). These possibilities should be further considered in terms of the very early mechanisms responsible for peptide bond formation. However, because we must consider the modern ribosome, we cannot avoid consideration of RNA in the evolution of biological systems.

It's remarkable how mainstream scientific papers give to their naturalistic proposals a positive connotation, but without providing compelling evidence for their assertions.

a Guanosine triphosphate ( GTP) is a high energy nucleotide (not to be confused with nucleoside ) found in the cytoplasm or polymerised to form the guanine base. 17 It is a result of it's complex three dimensional structure and the variety of different chemical groups which it comprises of.
Guanosine-5'-triphosphate (GTP) is a purine nucleoside triphosphate. It is one of the building blocks needed for the synthesis of RNA during the transcription process. Its structure is similar to that of the guanine nucleobase, the only difference being that nucleotides like GTP have a ribose sugar and three phosphates, with the nucleobase attached to the 1' and the triphosphate moiety attached to the 5' carbons of the ribose. It also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of ATP, but more specific. It is used as a source of energy for protein synthesis and gluconeogenesis. GTP is essential to signal transduction , in particular with G-proteins , in second-messenger mechanisms where it is converted to guanosine diphosphate (GDP) through the action of GTPases . 16

Guanosine is a purine nucleoside comprising guanine attached to a ribose (ribofuranose) ring via a β-N9-glycosidic bond. Guanosine can be phosphorylated to become guanosine monophosphate (GMP), cyclic guanosine monophosphate (cGMP), guanosine diphosphate (GDP), and guanosine triphosphate (GTP). These forms play important roles in various biochemical processes such as synthesis of nucleic acids and proteins, photosynthesis, muscle contraction, and intracellular signal transduction (cGMP). 18

For the synthesis purines, following enzymes are required:

phosphoribosylamine-glycine ligase,
phosphoribosylglycinamide formyltransferase,
phosphoribosylformylglycinamidine synthase,
phosphoribosylformylglycinamidine cyclo-ligase, 20

Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA.
For scientists attempting to understand how the building blocks of RNA originated on Earth, guanine -- the G in the four-letter code of life -- has proven to be a particular challenge. While the other three bases of RNA -- adenine (A), cytosine (C) and uracil (U) -- could be created by heating a simple precursor compound in the presence of certain naturally occurring catalysts, guanine had not been observed as a product of the same reactions.

How could and would random events attach a phosphate group to the right position of a ribose molecule to provide the necessary chemical activity? And how would non-guided random events be able to attach the nucleic bases to the ribose? The coupling of a ribose with a nucleotide is the first step to form RNA, and even those engrossed in prebiotic research have difficulty envisioning that process, especially for purines and pyrimidines.”
The sugar found in the backbone of both DNA and RNA, ribose, has been particularly problematic, as the most prebiotically plausible chemical reaction schemes have typically yielded only a small amount of ribose mixed with a diverse assortment of other sugar molecules. 16

Glycosidic bond
The formation of nucleosides in abiotic conditions is a major hurdle in origin-of-life studies. The formamido pyrimidine-based syntheses are high regioselective, moderately stereoselective, multi-step, only apply to purines and afford a mixture of furanosides and pyranosides. The prebiotic worth of these syntheses is inversely proportional to the procedural complexities involved, requiring numerous concentration, purification and supplementation steps, designed to specifically overcome intermediate reactions bottlenecks. 21

Guanosine monophosphate (GMP)

b CCA is a terminal sequence required for the function of all tRNAs, is added to the 3' ends of tRNA molecules for which this terminal sequence is not encoded in the DNA. The enzyme that catalyzes the addition of CCA is atypical for an RNA polymerase in that it does not use a DNA template. A third type of processing is the modification of bases and ribose units of ribosomal RNAs. 6 CCA is added by the CCA-adding enzyme (Figure below).

Transfer RNA precursor processing.
The conversion of a yeast tRNA precursor into a mature tRNA requires the removal of a 14-nucleotide intron (yellow), the cleavage of a 59 leader (green), and the removal of UU and the attachment of CCA at the 39 end (red). In addition, several bases are modified.

Eukaryotic tRNAs are also heavily modified on base and ribose moieties these modifications are important for function. In contrast with prokaryotic tRNAs, many eukaryotic pre-tRNAs are also spliced by an endonuclease and a ligase to remove an intron.

tRNA nucleotidyltransferase adds the invariant CCA terminus to the tRNA 30-end, a central step in tRNA maturation.7

Protein synthesis takes place in cytosolic ribosomes, mitochondria (mitoribosomes), and in plants, the plastids (chloroplast ribosomes). Each of these compartments requires a complete set of functional tRNAs to carry out protein synthesis. The production of mature tRNAs requires processing and modification steps such as the addition of a 3’-terminal cytidine-cytidine-adenosine (CCA). Since no plant tRNA genes encode this particular sequence, a tRNA nucleotidyltransferase must add this sequence post-transcriptionally and therefore is present in all three compartments. 8

cEF-G (elongation factor G, historically known as translocase) is involved in protein translation. As a GTPase, EF-G catalyzes the movement (translocation) of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome. EF-G is made up of 704 amino acids that form 5 domains , labeled Domain I through Domain V. 9

d The joining of an amino acid to a tRNA molecule to form an aminoacyl-tRNA is catalyzed by a specific enzyme called an Aminoacyl tRNA synthetase 14. An aminoacyl-tRNA synthetase (aaRS or ARS), also called tRNA-ligase, is an enzyme that attaches the appropriate amino acid onto its tRNA. It does so by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. In humans, the 20 different types of aa-tRNA are made by the 20 different aminoacyl-tRNA synthetases, one for each amino acid of the genetic code. This is sometimes called "charging" or "loading" the tRNA with the amino acid. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code. Aminoacyl tRNA therefore plays an important role in RNA translation, the expression of genes to create proteins. 13

e A ternary complex is a protein complex containing three different molecules that are bound together. 15

6. Styer, Biochemistry, 8th. edition, page 870
12. Styer, Biochemistry, 8th. edition, page 36

Last edited by Otangelo on Sat Jan 02, 2021 12:09 am edited 61 times in total

The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit forms on the mRNA template either at the Shine-Dalgarno sequence (prokaryotes) or the 5' cap (eukaryotes). Translation begins at the initiating AUG on the mRNA, specifying methionine. The formation of peptide bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. Charged tRNAs enter the ribosomal A site, and their amino acid bonds with the amino acid at the P site. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a nonsense codon is encountered, a release factor binds and dissociates the components and frees the new protein. Folding of the protein occurs during and after translation.

Figure Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

Chloramphenicol would directly affect

Figure Tetracycline: a Chloramphenicol: c.

Molecular Basis of Inheritance | Study Notes | Class-12 Biology

✔ DNA is a long polymer of deoxyribonucleotides.

✔ The length of the DNA depends on the number of nucleotide base pair present in it.

✔ Bacteriophage ø𴢆 has 5386 nucleotides.

✔ Bacteriophage lambda has 48502 base pairs.

✔ Escherichia coli have 4.6 X 10 6 base pairs.

✔ Human genome (haploid) consists of 3.3 × 10 9 base pairs.

Structure of Polynucleotide Chain

✔ DNA and RNA are referred to as Polynucleotide chain

Nucleotide : Nucleoside + Phosphate moiety (linked via Phosphodiester bond)

Nucleoside : Pentose Sugar + Nitrogenous base (linked via the N-Glycisidic Bond)

✔ Pentose sugar is Ribose in RNA and Deoxyribose in DNA.

✔ Nitrogenous base are two types

o Purine (Adenine and Guanine)

o Pyrimidine (Cytosine, Thymine and Uracil)

✔ Uracil is found only in RNA in place of Thymine.

✔ Number of nucleotides joins together through 3’ – 5’ phosphodiester bond to form the polynucleotide chain.

✔ Polynucleotide chain has a free phosphate moiety at 5’ end of sugar, is referred to as 5’ end and a 3’-OH group on the other end called 3’ end.

Sugar and phosphate forms the backbone of polynucleotide chain.

Discovery of DNA and determination of its structure

✔ Friedrich Meischer: Discovered the DNA in 1869 and named it as ‘Nuclein’.

✔ Wilkins & Franklin : Produced the X-ray diffraction data for DNA structure.

✔ Watson & Crick : Proposed double helix structure model for DNA based on X-ray diffraction data.

✔ For a double stranded DNA (dsDNA), the ratio between Adenine and Thymine, and Guanine and Cytosine are constant and are equal to one.

Salient features of the Double-helix structure of DNA

✔ Two polynucleotide chains are coiled to form a double helix. Sugar-phosphate forms backbone for the helix. The bases project in wards to each other.

✔ The two chains have antiparallel polarity.

✔ The nitrogen bases of the two strands are paired by Hydrogen bonding. A purine always pairs with a pyrimidine. A = T and C Ξ G.

✔ The plane of one base pair stacks over the other. This provides stability to the helix along with hydrogen bonding.

✔ The helix is right handed.

o Pitch (distance parallel to helix that corresponds to one turn of 360 0 ) : 3.4 nm or 34A ̊ .

o Number of bases per turn: 10

o Distance between two adjacent base pair: 0.34 nm.

Central Dogma of Molecular Biology

✔ Proposed by Francis Crick.

✔ Talks about the direction of flow of genetic information.

Packaging of DNA Helix

✔ There is 6.6吆 9 bp per cell in mammals. Taking 0.34 nm as the distance between consecutive bp, the total length of DNA happens to be 2.2 meters. (6.6 X10 9 bp X 0.34 X10 -9 m=2.2 meters)

✔ 2.2 m of DNA is too large to be accommodated in the nucleus with a dimension of 10 𕒺 m.

Packaging of DNA in Prokaryotes (E.g. E. coli)

✔ Prokaryotes lack a well-defined nucleus.

✔ Genetic material is scattered in the cytoplasm.

Nucleoid : The region where DNA being negatively charged (due to phosphate moiety) is associated with protein that is positively charged.

Packaging of DNA in Eukaryotes

Histones : Positively charged, basic protein.

✔ Histones are rich in basic amino acids like Lysine and arginine that give it positive charge.

✔ Histone octamer: Unit of eight molecules of histone.

✔ DNA (negatively charged) wraps around histone (positively charged) to form nucleosomes.

✔ 1 nucleosome has approximately 200 bp of DNA.

✔ Nucleosomes in a chromatin resemble beads present on strings.

✔ Beads on string structure in chromatin are further packaged to form chromatin fibres, which further coil and condense to form chromosomes during metaphase.

Non-histone chromosomal (NHC) proteins − Additional set of proteins required for packaging of chromatin at higher level

Chromatin Types

o Loosely coiled region of chromatin.

o Transcriptionally active.

o Tightly coiled region of chromatin.

o Transcriptionally inactive.

The search for genetic material

Transforming Principle

✔ Experiments performed by Griffith on Streptococcus pneumonia.

S. pneumonia has two strains: R strain and S strain.

✔ Produces a smooth colony on culture plate.

✔ Produces a rough colony on culture plate.

✔ Produces a polysaccharide coat.

✔ Polysaccharide coat absent.

✔ Experiment performed by Griffith

✔ Live R strain in the presence of heat-killed S strain should not have killed the mouse.

✔ Somehow the bacteria produce virulence.

✔ This is because somehow the R strain bacteria are transformed by heat-killed S strain bacteria.

✔ The transformation must be due to transfer of genetic material.

Biochemical Nature of Transforming Material

✔ Avery, MacLeod and McCarty worked to determine the biochemical nature of ‘transforming principle’ in Griffith's experiment.

✔ They tried transforming the R cells to S cells by using biochemical (proteins, DNA, RNA, etc.) extracted from the S cell.

✔ They concluded that DNA is the genetic material, as only it could transform the bacterial strain.

DNA as the Genetic Material

✔ The proof came from the experiment performed by Hersey and Chase.

✔ They used bacteriophage-Virus that infects bacteria.

o Upon infection the bacteriophage injects its DNA to the host cell and gets integrated to the host genome and subsequently produces more viral particles using the host machineries.

✔ They experimented to find out whether protein or the DNA that entered the bacterial cell.

Hersey and Chase Experiment

✔ They grew some viruses on a medium that contained radioactive phosphorus ( 32 P) and some others on medium that contained radioactive sulphur ( 35 S).

✔ Media containing radioactive Phosphorous had radioactive DNA.

✔ Media containing radioactive sulphur had radioactive protein.

Infection : These radioactive bacteriophages were used to infect E. coli. Phage transfers the genetic material to the bacterial cell.

Blending : Viral coat were separated from the bacterial cell by agitating them in a blender.

Centrifugation : Viral particles were separated from the bacterial cell by spinning them in a centrifuge machine.

o Bacteria infected with phage with radioactive protein ( 35 S)

▪ No radioactivity detected in cell

▪ Radioactivity detected in the supernatant.

o Bacteria infected with phage with radioactive DNA ( 32 P)

▪ Radioactivity detected in cell

▪ No-radioactivity detected in the supernatant.

o The above observation concluded that it is the DNA that entered the bacteria from phage and not proteins.

o Hence, it was concluded that DNA is the genetic material and not the protein.

Properties of Genetic Material (DNA and RNA)

✔ Criteria for a biomolecule to be genetic material:

o It should be able to make copy of it self (Replicate).

o It should be structurally and chemically stable.

o It should all the scope for mutation (essential for the process of evolution).

o It should express itself following the Mendelian principles of inheritance.

Stability of RNA

✔ The 2′ OH group in RNA is present at every nucleotide makes RNA unstable and degradable.

✔ RNA also acts as catalyst (ribozyme), hence reactive.

✔ RNA mutates faster compared to DNA, as it is unstable.

Stability of DNA

✔ The complementarity of the two DNA strands provides stability to the molecule.

✔ Thymine instead of uracil in DNA provides additional stability.

✔ DNA being more stable chemically and structurally is the preferred nucleic acid for storage of genetic material.

✔ RNA was the first genetic material and essential life processes evolved around it.

✔ DNA has evolved from RNA with chemical modification that makes it more stable.

✔ During replication two strands of DNA separate and act as template for the synthesis of new DNA strand that are complementary.

Semiconservative DNA replication : After one complete replication cycle, each DNA molecule consists of a parental DNA strand and a newly synthesised strand.

Proof of Semiconservative nature (Meselson and Stahl’s Experiment):

✔ Experiment performed by Meselson and Stahl, on E. coli.

✔ They used heavier isotope of nitrogen 15 N in the media as the only nitrogen source for many generation

✔ 15 N gets incorporated into the newly synthesized DNA (heavy DNA).

✔ The E. coli then were allowed to grow on a medium with 14 N as the nitrogen source.

✔ Samples were taken at definite time intervals (20 minutes/one generation) as the cells multiplied and the DNA was extracted.

✔ Heavy DNA can be separated from the normal DNA by CsCl density gradient.

✔ Various samples were centrifuged independently using Cscl Density gradient centrifugation.

✔ The DNA that was extracted from the culture before transferring to the 15 N medium had a heavy density.

✔ DNA extracted from the culture one generation after the transfer from 15 N to 14 N had a hybrid or intermediate density.

✔ DNA extracted after another generation was composed of equal amounts of this hybrid DNA and of ‘light’ DNA.

✔ Form the above observations they concluded that DNA replication is semi-conservative in nature.

Mechanism of DNA replication

✔ The DNA replication occurs in the S phase of cell cycle.

✔ The main enzyme involved is the DNA dependent DNA polymerase.

Deoxyribonucleoside triphosphates (dNTPs) serve dual purposes:

o Provides energy the polymerisation reaction

✔ DNA replication begins at specific site called the origin of replication.

Replication fork : During replication the two DNA strand do not completely open up. Instead a small portion is opened up where replication occurs. This site is called replication fork.

✔ DNA polymerase polymerises the new strand only in one direction, i.e. 5’𔾷’.

Continuous/ leading strand : One of the DNA strand with polarity 3’𔾹’ that acts as template for the new strand synthesis, synthesises the strand continuously.

Discontinuous/ lagging strand : The other strand with polarity 5’𔾷’, acting as the template synthesizes the new strand in fragments (Okazaki fragments) that are later joined together by the enzyme DNA ligase.

✔ The process of formation of RNA from DNA is referred to as transcription.

✔ Only a segment of DNA from only one of the two strands participates in the process of transcription.

✔ Both strands are not copied during transcription because of the following:

o If both strands get transcribed at the same time then two RNA molecules with different sequences will be formed, and in turn if they code for protein, two different sequences of amino acid would be formed, which in turn give rise to two different proteins. Therefore, one DNA fragment would end up giving rise to two different proteins.

o Two RNA molecules so formed will be complementary to each other, hence would end up forming a double-stranded RNA leaving the entire process of transcription futile.

Transcriptional Unit

✔ A transcriptional unit has primarily three regions:

o Promoter − Marks the beginning of transcription RNA polymerase binding site

o Structural gene − Part of the DNA that is actually transcribed

o Terminator − Marks the end of transcription

Structural gene: Template/coding strand

✔ Template strand: DNA strand with polarity 3’𔾹’

✔ Coding strand: DNA strand with polarity 5’𔾷’

✔ The sequence of RNA is same with that of the Coding strand.

✔ All the reference point while defining a transcription unit is made with coding strand.

✔ The region of DNA where RNA polymerase binds.

✔ Located towards the 5’ end or upstream region of structural gene.

✔ Region of DNA that defines the end of transcription.

✔ Located towards the 3’ end or downstream region of the structural gene.

✔ This is the site where the termination factor (ρ factor) binds to the RNA polymerase.

Transcription Unit and the Gene

Gene : The DNA sequence which codes for tRNA or rRNA molecule.

Cistron : Segment of DNA that contains the genetic code for a single polypeptide.

✔ The structural genes could be of two types:

o Monocistronic (mostly in eukaryotes)

o Polycistronic (mostly in prokaryotes)

✔ Monocistronic genes have two parts:

o Exon : Sequences that code for a particular character and is expressed in a matured and processed mRNA.

o Intron : Interrupting sequences that do not appear in a mature and processed mRNA.

mRNA (messenger RNA) : It serves as a template for protein synthesis.

tRNA (transfer RNA) : It brings amino acids during translation and reads the genetic code. Possess the anti-codon.

rRNA (ribosomal RNA) : They play a structural and catalytic role during translation.

Process of Transcription:

✔ Transcription has three steps − initiation, elongation, and termination.

✔ RNA polymerase binds to promoter and start transcription.

✔ The process is catalysed by the DNA dependent RNA polymerase.

✔ It associates transiently with initiation-factor (σ) to initiate the process.

✔ RNA polymerase uses nucleoside triphosphates (NTPs) as substrate to polymerise the new strand following the rule of complementarity.

✔ On reaching the terminator region, the RNA polymerase associates with the termination factor (ρ).

✔ As a result of this the nascent RNA falls off, so also the RNA polymerase.

✔ This results into the termination of the process.

Prokaryotic Transcription

✔ mRNA does not require any processing to become active.

✔ As there is no separate nucleus in prokaryotes, translation can begin before transcription is completed.

✔ Thus, in prokaryotes, transcription and translation are coupled.

Eukaryotic Transcription

✔ Three RNA polymerases are present

o RNA polymerase I transcribes rRNA (28S, 18S and 5.8S)

o RNA polymerase II transcribes hnRNA (mRNA precursor).

o RNA polymerase III transcribes tRNA, snRNA, and 5s rRNA.

o Heterogeneous nuclear RNA (hnRNA) containts both introns and exons.

o RNA splicing: Introns are removed by this process and Exons are joined together.

o Capping: A methyl guanosine triphosphate residue is added to the 5′ end of hnRNA.

o Tailing: 200-300 adenylate residues are added to the 3′ end of hnRNA.

o Fully processed hnRNA, now called mRNA, that is transported out of the nucleus for translation.

✔ Genetic code that directs the sequence of amino acids during synthesis of proteins.

✔ Change in nucleic acid is responsible for change in amino acids in proteins.

George Gamow : Proposed that in order to code for all the 20 amino acids, the code should be made up of 3 nucleotides.

Codon : Sequence of three nucleotides that corresponds to a specific amino acid or stop signal during protein synthesis.

Har Gobind Khorana : gave rise to the checker-board for genetic code.

✔ The salient features of genetic code are as follows:

o The codon is triplet. 61 codons code for amino acids and 3 codons do not code for any amino acids (stop codons).

o Unambiguous: One codon codes for only one amino acid.

o Degenerate: Some amino acids are coded by more than one codon.

o No punctuations: The codon is read in mRNA in a contiguous fashion.

o Universal: One codon codes for the same amino acid in almost all the species.

o AUG has dual functions. It codes for Methionine and also acts as initiator codon.

Mutations and Genetic Code

✔ Mutations include insertions, deletions, and rearrangements.

✔ Mutation results in changed phenotype and diseases such as sickle cell anaemia. (Change Glu→ Val in gene coding for β-globin chain of haemoglobin).

✔ Insertion or deletion of a single base pair disturbs the entire reading frame in mRNA. Such mutations are called frameshift mutations.

✔ Frameshift mutations hold the proof of the fact that codon is triplet because if we insert three or multiple of three bases followed by the deletion of same number of bases, then the reading frame will remain unaltered.

tRNA-The Adapter Molecule

✔ tRNA is the adapter molecule that on one hand read the code and on other hand bind to specific amino acid.

Anticodon loop : It has bases complementary to the code

✔ Amino acid acceptor end: Contains amino acid binding site.

✔ tRNAs are specific for each amino acid.

Initiator tRNA : It carries formylated metheionine (f-met). It helps in initiation of translation.

✔ The secondary structure of tRNA looks like a clover-leaf.

✔ It is the process where polypeptide chains (proteins) are formed from a mRNA.

✔ Amino acids are polymerised (joined by peptide bond) to form a polypeptide.

✔ At first charging of tRNA (amino-acylation of tRNA) takes place. In this, amino acids are activated in the presence of ATP and are linked to their corresponding tRNA. This energetically favours the formation of peptide bond between two amino acids.

✔ Translation occurs in Ribosome. Ribosomes have 2 subunits: a large subunit and a small subunit.

✔ Smaller subunit comes in contact with mRNA to initiate the process of translation.

✔ Translational unit in an mRNA is the region flanked by start codon and stop codon.

✔ Untranslated regions (UTR) are the regions on mRNA that are not translated, but are required for efficient translation process. They are present before start codon (5′ UTR) or after stop codon (3′ UTR).

Initiation : Initiator tRNA recognises the start codon.

Elongation : The t-RNA-amino acid complexes bind to their corresponding codon on the mRNA and base pairing occurs between codon on mRNA and tRNA anticodon. tRNA moves from codon to codon on the mRNA and amino acids are added one by one.

Termination : Release factor binds to stop codon to terminate the translation.

Regulation of gene expression

✔ In eukaryotes, the regulation could be exerted at:

o transcriptional level (formation of primary transcript),

o processing level (regulation of splicing),

o transport of mRNA from nucleus to the cytoplasm,

✔ The metabolic, physiological or environmental conditions that regulate the expression of genes.

Prokaryotic regulation of gene expression

✔ Gene expression is regulated by controlling the rate of transcriptional initiation.

✔ The activity of RNA polymerase at a given promoter is regulated by accessory proteins. The accessory proteins affect the ability of a promoter to recognise start sites.

✔ A regulatory protein could be activator or repressor.

✔ Accessibility of promoter is also affected by operators. Operator is the region located adjacent to promoter.

Operon - a polycistronic structural gene is regulated by a common promoter and regulatory genes.

✔ Each operon has a specific operator and a specific repressor.

✔ Usually operator binds to a repressor protein.

✔ Lac operon was first described by Jacob and Monad.

✔ Bacteria generally prefer glucose as the carbon source to obtain energy.

✔ In conditions when lactose is the only source of carbon in the environment, the bacteria synthesises the β-galactosidase enzyme that breaks down the lactose into glucose and galactose.

✔ β-galactosidase (along with permease and transacetylase) that is required for lactose utilization is part of the lac operon.

✔ The lac operon consists of following gene:

o ‘i’ gene (inhibitor) : It codes for repressor of lac operon.

o ‘z’ gene (structural gene): It codes for β-galactosidase.

o ‘y’ gene (structural gene): It codes for permease, which increases the permeability of cell to lactose.

o ‘a’ gene (structural gene): It codes for transacetylase.

Inducer : lactose acts as inducer. It regulates switching on and off of the operon.

Lac operon: in Absence of Inducer

✔ The repressor is synthesised all-the-time from the ‘i’ gene.

✔ The repressor protein binds to the operator region and prevents RNA polymerase from transcribing the operon.

✔ In this condition the lactose cannot be metabolised (as gene z, y and a are not expressed).

Lac operon: in presence of Inducer

✔ The inducer, allolactose (an alternate form of lactose), when present binds to the repressor and inactivated it.

✔ This inactivated repressor is unable to inactivate RNA polymerase enzyme and z, y, and a genes synthesise their respective mRNA, which in turn gets translated to form β-galactosidase, permease, and transacetylase.

✔ In presence of all these enzymes, the metabolism of lactose proceeds in a normal manner.

✔ This kind of regulation of lac operon is referred to as negative regulation.

Human Genome Project

✔ Joint venture of US department of energy and National Institute of Health (NIH) later joined by Welcome Trust (UK)

✔ Launched in 1990, completed in 2003

✔ This project worked towards the determination of complete DNA sequence of humans.

✔ Human genome (genome refers to the total genes that are present in a human being) contains 3 × 10 9 base pairs.

✔ If the obtained sequences were to be stored in typed form in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to store the information of DNA sequence from a single human cell.

✔ This creates problem for the storage and retrieval, and analysis of data.

✔ To solve this problem high speed computational devices were used. (A branch of biology called, bioinformatics developed).

✔ Some of the important goals of HGP were as follows:

o Identify all the approximately 20,000-25,000 genes in human DNA

o Determine the sequences of the 3 billion chemical base pairs that make up human DNA

o Store this information in databases

o Improve tools for data analysis

o Transfer related technologies to other sectors, such as industries

o Address the ethical, legal, and social issues (ELSI) that may arise from the project.

✔ Genomes of many non-human models such as bacteria, yeast, Caenorhabditiselegans, Drosophila, plants (rice and Arabidopsis) have also been sequenced.

Methodologies used

✔ Two methods: identifying ESTs (Expressed sequence Tags) and sequence annotation

✔ ESTs: As the name suggests, this refers to the part of DNA that is expressed, i.e. transcribed, as mRNA and translated into proteins thereafter. It basically focuses on sequencing the part denoting a gene.

Annotation: In this approach, entire genome (coding + non-coding) is sequenced and later on function is assigned to each region in the genome.

Genome Sequencing

✔ DNA from the cells is isolated and is randomly broken into fragments of smaller sizes.

✔ These fragments are cloned into suitable host using vectors.

✔ Cloned fragments amplify in the host. Amplification facilitates an easy sequencing.

✔ Common vectors used: BAC (Bacterial artificial chromosomes) and YAC (Yeast artificial chromosomes)

✔ Common hosts: Bacteria and yeasts

✔ Automated sequencers are used to sequence these smaller fragments (Sanger sequencing).

✔ The sequences so obtained are arranged based on overlapping regions within them (alignment).

✔ Alignment of the sequences is also done automatically by computer programs.

✔ Then these sequences are annotated and assigned to each chromosome.

Genetic and physical maps on Genome

✔ This was generated using information on polymorphism of restriction endonuclease recognition sites, and some repetitive DNA sequences known as microsatellites.

Salient Features of Human Genome

✔ The human genome contains 3164.7 million nucleotide bases.

✔ The average gene consists of 3000 bases.

✔ The total number of genes is estimated at 30,000.

✔ The functions are unknown for over 50 % of the discovered genes.

✔ Less than 2% of the genome codes for proteins.

✔ Repeated sequences make up very large portion of the human genome.

✔ Repetitive sequences that are repeated many times thought to have no direct coding functions, but they shed light on chromosome structure, dynamics and evolution.

✔ Chromosome 1 has most genes (2968), and the Y has the fewest (231).

✔ Scientists have identified about 1.4 million locations where single-base DNA differences (SNPs – single nucleotide polymorphism) occur in humans.

✔ DNA fingerprinting is a method employed to compare the DNA sequences of any two individuals.

✔ Of the total base sequence present in humans, 99.9% in all human beings are identical. The remaining 0.1% differs from person to person and makes every individual unique.

✔ It is a really difficult and time-consuming task to sequence and compare all 3 × 10 9 bases in two individuals. So, instead of considering the entire genome, certain specific regions called repetitive DNA sequences are used for comparative study.

Basics of DNA fingerprinting

✔ In a density gradient centrifugation of bulk genomic DNA, most of the DNA formed a major peak, but the rest of the DNA formed a smaller peak called the Satellite DNA.

✔ Satellite DNA can be categorised as mini- or micro- depending on the following:

o Base composition (A:T and G:C rich)

o Number of repetitive units

✔ These sequences do not code for any protein.

✔ Polymorphism is variation at genetic level that arises due to mutation.

✔ Satellite DNA show high degree of polymorphism and form the basis of DNA fingerprinting.

✔ As the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes.

Polymorphisms arise normally in the non-coding sequences because a mutation in non-coding sequences does not affect an individual’s reproductive ability.

Technique of DNA fingerprinting

✔ The technique was developed by Alec Jeffreys.

Variable Number of Tandem Repeats (VNTR) is a type of satellite DNA that shows high degree of polymorphism.

✔ The various steps involved are as follows:

o Digestion of the DNA with the help of restriction endonuclease (RE).

o The digestion with RE creates DNA fragments which are then separated by gel electrophoresis.

o The separated DNA fragments are blotted (immobilised) onto synthetic membranes, such as nitrocellulose or nylon.

o Immobilised fragments are hybridised with a VNTR probe.

o Detection of hybridised DNA fragments by autoradiography.

✔ VNTRs vary in size from 0.1 to 20 kb.

✔ The autoradiogram, after hybridization with VNTR probe gives many bands of different sizes.

✔ These bands give a characteristic pattern for an individual DNA.

✔ They are different in each individual, except identical twins.

✔ Identification of the criminals.

✔ Population diversity determination.

✔ Determination of genetic diversity.

Download PDF Notes for Molecular Basis of Inheritance - Class-XII.