Synthesis of proteins modified

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  • 1. FUNCTION OF GENETIC MATERIAL A gene is a section of the DNA molecule which extends the length of the chromosome, more or less at its centre, and hence forms the core of a chromosome. The ultimate function of a gene in an individual is to control and influence its phenotype.
  • 2.  However between the gene and the ultimate phenotype of the individual there occur many complex events.
  • 3.  The main ideas to explain the mode by which genes are able to exercise their control on phenotypic expression has been hypothesized as follows: All biochemical processes in all organisms are controlled by genes. The biochemical processes proceed in series of individual stepwise reactions.
  • 4.  Each single reaction is controlled in a primary fashion by a single gene, so that a one-to- one correspondence exists between genes and biochemical reactions. Mutation of a single gene results only in an alteration in the ability of the cell to carry out a single primary chemical reaction.
  • 5.  Since all biochemical reactions are catalysed by enzymes then the ultimate product of a metabolic process may be considered to be affected by a stepwise succession of enzymes, each produced by a particular gene. This hypothesis is strongly supported by findings from various studies that certain hereditary human defects are associated with certain biochemical defects. The first known case of the genetic control of a specific chemical reaction was found in a rare metabolic disease in man called alcaptonuria.
  • 6.  The disease is characterised by the hardening and blackening of the cartilage of the bones and the blackening of urine on exposure to air. The blackening of urine is due to an accumulation of alkapton or homogentisic acid, which is an intermediate product of phenylalanine or tyrosine metabolism. The metabolic pathway of phenylalanine involves various intermediate steps each controlled by a specific enzyme, and hence by a specific gene.
  • 7.  In a normal person an enzyme is present that changes homogentisic acid to aceto-acetic acid, and pyruvic acid which are clear in urine. An alcaptonuric individual, however, lacks this enzyme, hence homogentisic acid accumulates in abnormal amounts in urine.
  • 8.  Apparently alcaptonuria is inherited as a recessive trait, but the condition is particularly manifested when alcaptonuric compounds such as phenylalanine and tyrosine are fed.
  • 9.  In phenyl-ketonuria (an imbecility disease) the affected individual lacks the enzyme phenylalanine hydrolase which is necessary for normal metabolism of phenylalanine to tyrosine. This leads to the accumulation of abnormal metabolites (including phenyl-pyruvic acid) in body tissues leading to idiocy.
  • 10.  Albinism is due to the lack of the enzyme tyrosinase which converts tyrosine into melanin. Thus when tyrosinase is not active, no pigment is formed in the individual.
  • 11.  The sickle-cell condition is due to the substitution of glutamic acid by valine in one position on the ß-polypeptide chain of haemoglobin protein molecule.
  • 12. αβ
  • 13.  Similar findings have been demonstrated in many organisms. On the basis of findings from numerous studies it has been established that a mutation in any of the genes controlling a metabolic pathway would lead to a blockade of the pathway.
  • 14.  The cause of the block is usually localized to an impaired function of the enzyme that is usually active at that particular metabolic step. However, all other subsequent steps may also be affected.
  • 15. Proteins and Protein Derivatives Proteins are large molecules consisting of amino acids linked together by peptide bonds that connect the carboxyl group of one amino acid with the amino group of another through loss of a water molecule.
  • 16.  Not only do different proteins differ with respect to their molecular size, but also with respect to the kinds of amino acids composing the proteins as well as the sequence of the amino acids, their relationship and frequency. Thus although only about twenty amino acids constitute most proteins the number of different kinds of proteins that can be formed is immensely large.
  • 17. - COOH
  • 18.
  • 19.  The amino acids are added one at a time in the polymerization of proteins. i.e. the assembly of proteins proceeds step by step and in only one chemical direction. Protein synthesis begins at the amino (-NH2) terminus and continues through to the carbocyl (-COOH) terminus.
  • 20.  In this way polypeptide chains consisting of many amino acid units are formed.
  • 21.  One or several polypeptide chains may constitute a protein molecule.
  • 22.  Although the theoretical number of amino acids is limitless only about 20 different acids are used in making proteins.
  • 23. Function of Proteins and their Derivatives Proteins are the active working components of cellular machinery. As a matter of fact, except for its water content, the major part of animal tissue is composed of protein The skeleton of animal body is composed of a protein matrix on which mineral compounds are deposited.
  • 24.  Muscles, cartilage, and many other body tissues are composed largely of proteins. Furthermore, the enzymes which play a major role in various metabolic processes including digestion, cell respiration, synthesis, etc, are essentially proteins.
  • 25.  Haemoglobin, a constituent of red blood cells which is responsible for oxygen supply to body cells and carbon dioxide transport to the lungs, is a protein.
  • 26.  The various antibodies responsible for body defense are essentially proteins. The hormones which play regulatory roles in various physiological processes in the animal body are all either proteins or protein derivatives.
  • 27. GENETIC CONTROL OF PROTEINSThe DNA molecule and Protein Synthesis It is well accepted now that the DNA controls the expression of characters in organisms through determination of the sequence of amino acids in polypeptide chains, and hence through determination of the secondary, tertiary and quarternally structures of the chains, which in turn determine the biochemical properties of the proteins.
  • 28.  i.e. the nucleotide sequence in the DNA molecule forms a sequence of codes which determine the order in which the amino acids will be linked together to form a protein. In turn the the linear order of amino acids in a protein molecule determines the function of the protein.
  • 29.  This proposition is especially appealing since both the DNA and the polypeptide chains are linear structures. While the DNA is composed of a linear sequence of nucleotides, the polypeptide chain consists of a linear sequence of amino acids Therefore it should be expected that a linear sequence of nucleotides should determine a specific linear sequence of amino acids.
  • 30.  Furthermore, a mutational change in a particular position of the nucleotide sequence should produce a change in a corresponding linear position in the amino acid sequence. Findings from various studies have shown that most gene mutations cause single amino acid substitutions.
  • 31.  Amino acid substitution on a polypeptide chain may have far-reaching effects on the organism carrying the mutant gene because the secondary and tertiary structure of the protein in which amino-acid substitution has occurred, and hence its biochemical properties may be completely altered. The function of some proteins is so sensitive that any change in primary amino acid structure of the molecules leads to observable phenotypic effects.
  • 32.  However such sensitivity does not exist for all proteins. i.e. some proteins seem to be completely functional despite significant changes in their amino acid sequence.
  • 33.  Since the linear order of amino acids in a protein molecule determines the function of the protein it is important that the mechanism for ensuring the order be very accurate and precise. The design and function of the protein synthesizing apparatus is similar in all cells of an organism and in all organisms.
  • 34.  Not only do different proteins differ with respect to their molecular size, but also with respect to the kinds of amino acids composing the proteins as well as the sequence of the amino acids, relationship and frequency. Thus although only about twenty amino acids constitute most proteins the number of different kinds of proteins that can be formed is immensely large.
  • 35. TRANSFER OF GENETIC INFORMATION By the early forties it had been established that while DNA was always confined only within the nucleus RNA existed both in the nucleus and in the cytoplasm.
  • 36.  These findings led to the proposition that RNA might be responsible for protein synthesis. It was further observed that RNA occurred in much larger amounts in high protein producing cells (e.g. liver and pancreas cells) than in low protein producing cells (e.g. kidney, heart, and lung cells).
  • 37.  The high protein producing cells had specific cytoplasmic areas that stained densely with basic dyes and absorbed ultraviolent radiation at a wavelength similar to nucleic acids. Also the enzyme that breaks down RNA (i.e. ribonuclease) caused a termination in protein synthesis and also removed the dark staining areas in the cytoplasm.
  • 38.  Later when methods of separating cellular contents (or organelles) by lysis of the cells followed by centrifussion, had been developed it was shown that most of the RNA was contained in the microsomes. Using labelling techniques with radioactive material it was shown that the labelled amino acids were rapidly assimilated in the microsomes, and that the acids were connected together by peptide bonds and incorporated into proteins.
  • 39.  Later the microsomal fraction was shown to consist of granules called ribosomes, associated with larger membranes called endoplasmic reticulae. The ribosomes were shown to contain most of the RNA and to perform protein synthesis. Further studies demonstrated that the ribosomes were a protein factory in themselves.
  • 40.  Today it is ribosomes are known to be complex intra-cellular structures composed of individual RNA molecules (rRNA) and more than 50 types of proteins, all organized into two sub-units, a large sub-unit and a smaller one. A ribosomal unit consists 40-60% ribosomal RNA (rRNA) and the rest is protein. Both the proteins and RNA molecules differ in the two sub-units, the large sub-unit possessing a large rRNA molecule, and the smaller sub-unit possessing a small rRNA molecule.
  • 41.  Further work pointed to a special form of RNA (i.e. the messenger RNA or mRNA) as carrier of genetic message from the gene located inside the nucleus of the cell to the surrounding cytoplasm where many of the proteins are synthesized.
  • 42.  As a result of numerous experimental findings the process of protein synthesis has now been well elucidated. The process is known to involve three kids of RNA which play cooperative roles in linking amino acids together in the correct linear arrangement.
  • 43.  Messenger RNA (mRNA) encodes genetic information that is copied from DNA. The copying (transcription) of mRNA from a DNA strand is achieved through the enzymatic action of RNA polymerase. The information is in the form of a sequence of bases that specifies a sequence of amino acids. The messenger RNA (mRNA) arranges itself on an unoccupied ribosome.
  • 44.  Another form of RNA, i.e. the ribosomal RNA (rRNA) combines with many different proteins to form ribosomes which provide binding sites for all the interacting molecules necessary for protein synthesis. Yet another form of RNA, i.e. the transfer RNA (tRNA) decodes (translates) the base sequence of the mRNA into the amino acid sequence of a protein.
  • 45.  The message for incorporation of amino acids into proteins resides solely in the nucleotide configuration of tRNA. tRNA molecules are short molecules about 70-80 nucleotides long and are of different types. Each type is able to recognize one or more of the several codons that can specify the same amino acid.
  • 46.  The transfer RNA (tRNA) seems to have a large portion of its structure in the form of a double helix, and also contains a number of rare bases such as pseudouridine and inosine, as well as some normal bases to which methyl groups have been added.
  • 47.  Some of the unusual nucleotides are unable to form hydrogen bonds with other bases and therefore produce looped sections in which the double helical structure of tRNA is interrupted. This gives the backbone of the tRNA structure a stem-loop appearance resembling a clover leaf.
  • 48.  The tRNA performs its function by: Picking a specific amino acid from the medium and carrying it to the mRNA; Attaching itself to the ribosome in accord with the sequence of nucleotide bases specified by mRNA. Protein formation then proceeds by linking the amino acids carried by neighbouring tRNA molecules.
  • 49.  The translation of nucleotide sequence on mRNA into a particular amino acid sequence is achieved with the help of ribosomes.
  • 50. The Protein Synthesis Process First the amino acids are activated through their attachment to adenosine tryphosphate (ATP), to form highly reactive amino-acyl-phosphate-adenyl groups. The enzymes involved in the formation of these groups are usually highly specific to particular amino acids.
  • 51.  Thus each of the twenty amino acids has its own activating enzyme or enzymes. Secondly free-floating transfer RNA molecules (tRNA) become attached to the amino acids and then transfer them to the ribosomes.
  • 52.  Again here there is a high degree of specificity between the tRNA molecules and the amino acids so that a certain type of tRNA would attach to only a particular amino acid. After the attachment of an amino acid molecule to a tRNA molecule the adenyl group is freed and the amino-acyl-tRNA travels to the ribosome, where a messenger RNA (mRNA) has been attached.
  • 53.  The messenger RNA serves as a template for the interconnection of different amino acids that are carried to the template by transfer RNA (tRNA) molecules to form a polypeptide chain which, either singly or together with other similar chains would constitute protein.
  • 54.  After the formation of a polypeptide chain has been completed both the polypeptide chain and the messenger RNA are detached from the ribosome which then becomes free to pick up a new messenger RNA.
  • 55.  The clover leaf-like structure consists of four base-paired stems and three loops, i.e. the didryuridine (D-loop), the anti-codon loop, and the TψGG loop.
  • 56.  The anti-codon loop contains three nucleotides that can form base pairs with the nucleotides of a specific codon of the mRNA. The three nucleotides in tRNA are called the anti-codon. They are complementary (not identical) to the three nucleotides in the mRNA codon.
  • 57.  Part of the ribonucleotide sequence of tRNA is added after it comes off the DNA molecule template.
  • 58.  The addition consists of an identical sequence of three nucleotides (A-C-C) which are attached to all the different tRNA molecules by a set of specific enzymes. One of the nucleotides in this terminal sequence (i.e. adenine) serves as the point of attachment to which a single amino acid is covalently bonded by a particular amino-acid activating enzyme.
  • 59. Activation of tRNA There are at least 20 amino acid-specific enzymes that recognize amino acids and their compatible (or cognate) tRNAs. Each enzyme can attach one amino acid molecule to the end of a cognate (appropriate) tRNA. A given enzyme is capable of recognizing different tRNAs for the same amino acid.
  • 60.  These enzymes are called amino acyl-tRNA synthetases. The amino acid is linked to the free 3 hydroxyl group of the ribose of the terminal nucleotide of the tRNA (adenosine).
  • 61.  The reaction is:1. Enzyme + amino acid + ATP ──── > enzyme-amino-acyl-AMP + Inorganic phosphate2. tRNA + enzyme – amino-acyl-AMP ──> amino-acyl-tRNA+AMP+enzyme.
  • 62. Summary Amino acid + tRNA + enzyme + ATP ────> Aminoacyl-tRNA + Enzyme + AMP + InorgPhos AA + tRNA+ATP ────> AA-tRNA+AMP + Inorganic Pyrophosphate.
  • 63.  The amino acid residue is said to have become activated and the tRNA is said to have become amino-acylated. The overall process releases AMP and inorganic pyrophosphate.
  • 64.  The basis of the specificity between a tRNA molecule and its cognate tRNA synthetase is probably due to their three dimensional structures. The fact that one enzyme can add the same amino acid to different tRNAs with different anti-codons suggests that the respective tRNAs must contain similar binding sites for the synthetase.
  • 65. • Three nucleotides of each tRNA molecule are used for coding purposes to pair with the triplet sequences of messenger RNA (mRNA).
  • 66.  The set of three nucleotides which pair with a particular triplet on the mRNA is called an anticodon. The location of the anti-codon is probably in one of the exposed positions on tRNA molecule, probably in one of the unpaired loops.
  • 67.  A tRNA molecule carrying a specific amino acid aligns itself on the ribosome, pairing its anticodon with the codon of mRNA. Next, a new mRNA codon is brought into position and a new tRNA molecule bearing an amino acid is positioned next to the previous tRNA.
  • 68.  Then the amino acid carried by the previous tRNA molecule is removed and linked through a peptide bond to the amino acid carried by the second tRNA molecule with the help of some enzymes and the energy rich molecule guanosine triphsphate (GTP).
  • 69.  Having lost its amino acid the previous tRNA molecule is released from the ribosome. In subsequent steps the linked amino acid chain is transferred to new tRNA molecules which have been attached to the ribosome. When the peptide chain is completed the ribosome detaches from the mRNA and the polypeptide chain is released from the last tRNA.
  • 70.  Also the last tRNA molecule is released from the ribosome.
  • 71.  The attachment of amino acids to their cognate tRNAs is a very critical stage in protein synthesis because once the tRNAs are loaded with the correct amino acids the accuracy of protein synthesis depends only on the base pairing between anti-codons on the tRNAs and the codons on the mRNA. A tRNA specific to a particular amino acid is designated as tRNA-AA where AA is the amino acid concerned.
  • 72.  If an amino acid residue which is already attached to its cognate tRNA is chemically changed into some other amino acid residue, the altered amino acid will still be added to the growing chain at the position where the cognate tRNA for the original amino acid would add it.
  • 73.  The ribosome is important for proper pairing between the three nucleotides constituting the anti-codon of the tRNA and the three nucleotides constituting the codon of the mRNA, and hence for the stabilization of trinucleotide attachment between mRNA and tNRA, otherwise such attachment would not be sufficiently strong or stable to permit the amino acids carried by tRNA to become linked together in peptide formation.
  • 74. Role of Ribosomes The critical function of protein synthesis would be very slow if the interacting components had to react in free solution since simultaneous collisions between the necessary components of the reaction would be rare.
  • 75.  Instead the mRNA with its encoded information and the individual tRNAs already loaded with their correct amino acids are brought together by their mutual binding to ribosomes. Thus the most important role played by the ribosome is to bind reversibly with both mRNA and tRNA.
  • 76. Sequence and rate of protein synthesis Experimental evidence indicates that protein synthesis occurs sequentially from one particular end of the chain to the other. It appears that growth starts at the amino end (N-terminal) of the polypeptide chain and continues towards the carboxyl end (C- terminal).
  • 77.  There is evidence suggesting that the sequential order of polypeptide synthesis follows the order of synthesis of the mRNA molecule itself. i.e. protein synthesis may begin even before synthesis of the mRNA is completed through the attachment of ribosomes to mRNA chain as it is coming off the DNA template.
  • 78.  The length of a polypeptide chain translated on a particular mRNA may not necessarily correspond with the nucleotide length of the mRNA molecule. Instead, several polypeptide chains may be translated on different sections of a mRNA molecule.
  • 79. SYNTHESIS OF PROTEINSRules for synthesis of proteinsProteins are made up of a limited number ofdifferent amino acids.Although the theoretical number of amino acidsis limitless only about 20 different acids are usedin making proteins.
  • 80. Protein Synthesis While DNA directs the synthesis of RNA, which in turn directs the synthesis of protein, special proteins catalyse the synthesis of both RNA and DNA. i.e. there is a cyclic flow of information in the cell.• DNA────>RNA ───>Protein• │ │ │• └───────┴─────────┘
  • 81.  Of the 64 possible codons under the 3-base code model only 3 do not specify amino acids. Since there are 61 codons for 20 amino acids many amino acids are coded by more than one codon.
  • 82.  Occasionally the DNA sequence may contain overlapping information still in a triplet code. Since it is possible to shift the reading frame for any set of triplets by moving the starting point for translation either one or two bases in either direction, two or three different amino acid sequences can be encoded by the same region of the nucleic acid chain.
  • 83.  Overlapping triplets read in two different frames - although the mRNA is the same sequence in both lines the sequence of amino acids coded in the region are very different.
  • 84.  The different codons for a given amino acid are said to be synonymous and the code itself is said to be degenerate - meaning that it contains redundancies. Since each triplet codes for only one amino acid there is no ambiguity in the translation of amino acids, except for GUG which apart from coding for the amino acid valine, may occasionally also code for methiomine.
  • 85.  AUG is the most common initiator or start codon specifying the amino acid methionine, while UAA, UAG and UGA act as termination codons. All protein chains in prokaryotic and eukaryotic cells begin with methionine.
  • 86.  The three codons UAA, UAG, and UGA do not specify any amino acids, and hence constitute termination (top) signals at the ends of protein chains. Therefore a precise linear arrangement of nucleotides grouped into triplets in the mRNA specifies, not only the linear sequence of amino acids in a protein, but also signals to ribosomes where to start and stop synthesis of a protein chain.
  • 87. Summary of degeneracy of codes.Amino acids Coded Codes per amino acid No. of codesArg, Leu and Ser 6 codes each x 3 = 18Ala, Gly, Pro, Thr and 4 codes each x 5 = 20ValIle 3 codes x 1 = 3Asn, Asp, Cys, Gln, 2 codes each x 9 = 18Glu, His, Lys, Phe, andTyrMeth and Trp 1 code each x 2 = 2Total number of codes 61for amino acidesNumber of codes not 3coding for amino acids
  • 88.  In the synthesis of a polypeptide chain the protein synthesizing system uses the tRNA to translate or adapt the information in each mRNA code word so that the appropriate amino acid is added to the chain. The adaptor molecule must recognize First, a codon in mRNA Second, an amino acid matching the codon
  • 89.  The adaptor function is performed by a tRNA molecule to which an amino acid molecule is attached at one end to form an aminoacyl- tRNA complex. The correct amino acyl-tRNA molecule binds to the codon on the mRNA strand and transfers its attached amino acid to the polypeptide chain growing there.
  • 90.  The structure of a tRNA molecule always ends in CCA. The amino acid is attached to the 3 hydroxyl group of the terminal nucleotide (i.e. adenosine).
  • 91.  In solution the tRNA molecules are folded into three dimensional structures. The backbone of the structure is a stem-loop structure resembling a clover leaf. The four stems are stabilized by base pairing.
  • 92.  Three of the four stems end in loops. The stem-loop structure is then folded into an L-shaped three-dimensional form. Hydrogen bonds help to maintain the molecules shape.
  • 93.  The tRNA bases are highly modified after tRNA is synthesized. The most frequent modification is the addition of a methyl group to specific bases.
  • 94.  Most tRNA molecules are synthesized with a four-base sequence of UψCG near the middle of the molecule. The first U-nucleotide is methylated to become a thymine (thymidine) nucleotide while the (uridine) U-nucleotide is rearranged into a pseudo uracil nucleotide in which the sugar is attached to a carbon instead of to a nitrogen.
  • 95.  These modifications produce a characteristic TψCG segment which is localed in an unpaired region at about the same position in nearly all tRNAS. A clover leaf-like structure consisting of four- base-paired stems and 3 loops - the didrydrouridine loop (D-loop), the anti-codon loop, and the TψCG loop.
  • 96.  Although the exact role of the tRNA modifications is not yet well understood the fact that certain sites on the tRNA structure are frequently modified in similar ways suggests that these sites have a common role in protein synthesis. The constant features are the D loop, the TψCG loop, and the anti-codon loop.
  • 97.  If perfect base pairing was required for codon-anti- codon pairing 61 different tRNA types (one for each codon) would have been necessary. But this is not the case. Rather, tRNA molecules with same anti-codon sequence are capable of recognizing more than one codon corresponding to a particular amino acid. This is possible due to wobble (or non-standard) base pairing between the third position of the codon and its partner in the anticodon. Certain combinations of two bases form interactions
  • 98.  This is possible due to wobble (or non- standard) base pairing between the third position of the codon and its partner in the anti-codon. Certain combinations of two bases form interactions For example A-U, G-C and several other combinations of two bases form interactions that are stable enough to allow codon recognition in the wobble position.
  • 99.  e.g. whereas the condon (5)UUU(3) in mRNA calls for phenylalanine-tRNA (Phe-tRNA Phe) the anti-codon in the Phe-tRNA-Phe could be either (3) AAA(5), (3)AAG)5) or (3) AAI(5). Inosine modified nucleoside (base) in which amine group of guanine has been substituted by a hydrogen atom) - a guanosine analogue that lacks an amino group at the No. 2 carbon position.
  • 100.  This is because bonds between U and G or between U and I in the wobble position No. 1 tRNA with inosine in the wobble position can decode three different codons. It is hyphothesized that the effect of the wobble in the third position is to speed up protein synthesis by the use of alternative tRNAs.
  • 101. Protein Synthesis The process of protein synthesis may be looked at in three stages: i.e. Initiation; Elongation and Termination. Each of these processes involves distinct biochemical events.
  • 102. Initiation It seems that the AUG codon of the mRNA is the initiation signal for polypeptide growth. This codon codes for methionine. Thus the first event of the initiation stage in the synthesis of any protein is the attachment of a free methionine molecule to the end of a tRNA met with the help of methionly-tRNA met synthetase.
  • 103.  The Met-tRNA Met so formed together with a molecule of GTP and the smaller ribosomal submit bind to the mRNA (with the help of initiation proteins- initiation factors) at a specific site near the AUG initiation code. Note that although there may be AUG codons in other places along the mRNA molecule, protein synthesis always begins at the correct AUG near the ribosomal binding sites. Translation then proceeds in the 5 ── 3 direction along the mRNA.
  • 104.  It seems that the recognition of AUG initiation sites is due to the high affinity for ribosomes by the mRNA base sequences just preceding the codons. An initiation factor first binds a GTP molecule and a molecule of Met-tRNA Met to form a complex which then binds to mRNA and the small ribosomal submit.
  • 105.  Other initiation factors then joint to make an initiation complex. These processes position the Met-tRNA met correctly at the AUG initiation code.
  • 106. Summary Met-tRNA +GTP+Ribosome + mRNA initiation factor ────> Met-tRNA-Ribosome-mRNA + initiation factor + inorganic phosphate
  • 107.  After the complex of Met-tRNAMet, GTP and the small ribosomal submit is correctly bound to the mRNA at the initiation site the large ribosomal submit joins the complex. This is followed by the hydrolysis of the GTP to GDP and inorganic phosphate, and the detachment of the initiation factors from the complex, leaving the Met-tRNAMet bound at the P site of the large ribosomal submit.
  • 108. Elongation A second amino acid that is correctly bound to its cognate tRNA is then brought into the second binding site (the A site) on the ribosome which positions the second tRNA at the appropriate codon of the mRNA. A peptide bond is then formed between the carboxyl group of the Met-tRNA Met and the amino group of the incoming aminoacyl-tRNA- AA2.
  • 109.  The tRNA-Met then vacates the P binding site of the ribosome into the medium, leaving behind the methionyl-aminoacyl-tRNA-AA2 (the peptidyl-tRNA-AA2) on the ribosome. In the meantime the peptidyl-tRNA-AA2 vacates the A site to the P site. The cycle is repeated for the addition of each amino acid, until all the amino acids encoded by the mRNA have been added.
  • 110.  In each translation step the ribosome and its attached peptidyl-tRNA move three nucleotides closer to the 3 end of the mRNA. i.e. advance one colon on the mRNA. This movement is probably achieved through the change in the configuration of some proteins of ribosome or in the configuration of RNA thus propelling the mRNA through the ribosome. The energy of GTP is probably used in the propulsion.
  • 111.  Since some of the hydrogen bonds existing in rRNA are between distant nucleotides the breakage and restitution of these bonds might be responsible for contraction and relaxation cycles which cause the folding of the ribosome to change, thus causing translation of the ribosome to occur. Note: The major role of the ribosome is to offer binding sites to amino-acyl- tRNA in such a way that the correct codon-anti-codon match is made.
  • 112. Termination When the UAG or UGA or UAA codon is encountered on the mRNA the protein termination factors cause the peptidly-tRNA complex to be hydrolysed and released from the ribosome and the complex splits instantaneously into an uncharged tRNA molecule and newly completed protein chain. After releasing its peptidyl-tRNA the ribosome disintegrates from the mRNA and divides into its two submits.
  • 113.  Experimental evidence has shown that a segment of about 35 amino acids long of the protein chain being synthesized is embedded within the ribosome structure at any time before the synthesis of the chain is completed. Therefore the chain starts to emerge from the ribosome only after it has grown more than 35 amino acids long. The protein secreted from the cell may go directly through the cell membrane, suggesting that the exit site on the ribosome may be bound to the cell membrane.
  • 114. Suppression of non-sense mutations Since UGA, UAA and UAG normally code for chain termination a mutation in a gene could produce an abnormal termination signals, causing the translation apparatus to stop too soon. This type of mutation is called a non-sense mutation. It is to be distinguished from a mis-sense mutation which would cause an amino acid to be substituted for another.
  • 115. • The chain terminating mutations on the mRNA are correctable by other mutations on the tRNA. These are called suppression mutations. Suppressor mutations cause the reading of the chain-terminating codon on the mRNA to be as a codon for an amino acid. This is brought by a mutation in the anti-codon of a tRNA leading to the production of a low frequency of misinterpretation of stop signals.
  • 116.  This would allow chain synthesis to continue. Due to the existence of suppressor tRNAs the 3 ends of coding regions in mRNA often contain more than one stop codons within a short stretch, giving the protein synthesis a fail-safe mechanism.
  • 117.  Each chain has a specific starting point, and growth proceeds in one direction to a fixed terminus. There are elaborate cellular mechanisms for starting and stopping the process correctly. The primary synthetic product is usually modified. The functional form of a protein molecule is rarely the same length as the initially synthesised form.
  • 118.  For example methyl groups can be added to specific sites of proteins. Also phosphate groups and a wide variety of polysaccharides can be added to proteins.
  • 119.  The original chain is often inactive or incomplete. Through the action of enzymes the original chain is trimmed down, linked to another chain, or even cut apart and reassembled from selected pieces to make a fully active chain. Primary chains may also undergo certain chemical additions either during their formation or after synthesis is complete.
  • 120.  Findings from studies with lower forms of life have indicated the existence of a close linkage of genes controlling the production of enzymes for a particular metabolic pathway. However such a correspondence between the sequence of genes and that of enzymes catalysing steps of a metabolic pathway is lacking in higher forms of life.