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Lecture 16

BCH2011: Textbook summary - Lecture 16

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LECTURE 16 Protein Biosynthesis Takes Place in Five Stages: Step 1: Activation of Amino Acids For the synthesis of a polypeptide with a defined sequence, two fundamental chemical requirements must be met: - The carboxyl group of each amino acid must be activated to facilitate formation of a peptide bond - A link must be established between each new amino acid and the information in the mRNA that encodes it. Both of these requirements are met by attaching the amino acid to a tRNA in the first stage of protein synthesis. Attaching the right amino acid to the right tRNA is critical. This reaction takes place in the cytosol, not the ribosome. When attached to their amino acid (aminoacylated) the tRNAs are said to be ‘charged’. Aminoacyl-tRNA Synthetases Attach the Correct Amino Acids to their tRNAs: During the first stage of protein synthesis, taking place in the cytosol, aminoacyl- tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each enzyme is specific for one amino acid and one or more corresponding tRNAs. Most organisms have one aminoacyl-tRNA synthetase for each amino acid. For amino acids with two or more corresponding tRNAs, the same enzyme usually aminoacylates all of them. Aminoacylation of tRNA by aminoacyl-tRNA synthetases: Step 1 is the formation of an aminoacyl adenylate, which remains bound to the active site. In the second step, the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases. For class I enzymes, the aminoacyl group is transferred initially to the 2’- hydroxyl group of the 3’- terminal A residue, then to the 3’- hydroxyl group by a transesterification reaction. For class II enzymes, the aminoacyl group is transferred directly to the 3’- hydroxyl group of the terminal adenylate. Proofreading by Aminoacyl-tRNA Synthetases: The aminoacylation of tRNA accomplishes two ends: - It activates an amino acid for peptide bond formation It ensures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. Interaction between an Aminoacyl-tRNA Synthetase and a tRNA: An individual Aminoacyl-tRNA synthetase must be specific not only for a single amino acid but for certain tRNAs as well. The interaction between Aminoacyl- tRNA synthetase and tRNA has been referred to as the ‘second genetic code’, reflecting its critical role in maintaining the accuracy of protein synthesis. Step 2: Initiation The mRNA bearing the code for the polypeptide to be synthesized binds to the smaller of two ribosomal subunits and to the initiating aminoacyl-tRNA. The large ribosomal subunit then binds to form an initiation complex. The initiating aminocyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning of the polypeptide. This process, which requires GTP, is promoted by cytosolic proteins called initiation factors. A Specific Amino Acid Initiates Protein Synthesis: Protein synthesis begins at the amino-terminal end and proceeds by the stepwise addition of amino acids to the carboxyl-terminal end of the growing polypeptide. The AUG initiation codon thus specifies an amino-terminal methionine residue. Although methionine has only one codon, (5’) AUG, all organisms have 2 tRNAs for methionine. One is used exclusively whn (5’) AUG is the initiation codon for protein synthesis. The other is used to code for a Met residue in an internal position in a polypeptide. The Three Steps of Initiation: In step 1, the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The mRNA then binds to the 30S subunit. The initiating (5’) AUG is guided to its correct position in the mRNA. Bacterial ribosomes have 3 sites that bind tRNAs: - The aminoacyl (A) site - The Peptidyl (P) site - The exit (E) site The A and P sites bind to aminoacyl-tRNAs, whereas the E site blinds only to uncharged tRNAS that have completed their task on the ribosome. Both the 30S and 50S subunits contribute to the characteristics of the A and P sites, whereas the E site is largely confined to the 50 subunit. The initiating (5’) AUG is positioned at the P site, the only site to which fMet-tRNA fMetcan bind. The fMet-tRNA fMeis the only aminoacyl-tRNA that binds to the P site; during the subsequent elongation stage, all other incoming aminoacyl-tRNAs (including the fMet-tRNA Metthat binds to interior AUG codons) bind first to the A site and only subsequently to the P and E sites. The E site is the site from which the ‘uncharged’ tRNAs leave during elongation. Factor IF-1 binds at the A site and prevents tRNA binding at this site during initiation. In step 2 of the initiation process, the complex consisting of 30S ribosomal subunit, IF-3, and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNA fMe. The anticodon of this tRNA now pairs correctly with the mRNA’s initiation codon. In step 3, this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolysed to GDP and Pi, which are released from the complex. All three initiation factors depart from the ribosome at this point. Initiation Complex: Completion of these 3 steps produces a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNA fMe. The fMet correct binding of fMet-tRNA to the P site in the complete 70S initiation complex is assured by at least 3 points of recognition and attachment: the codon- anticodon interaction involving the initiation AUG fixed in the P site, interaction between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA, and binding interactions between the ribosomal P site and the fMet-tRNA fMetThe initiation complex is now ready for elongation. Step 3: Elongation The nascent polypeptide is lengthened by covalent attachment of successive amino acid units, each carried to the ribosome and correctly positioned by its tRNA, which base
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