Protein Synthesis II

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• published: 20 Oct 2010
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This course is part of a series taught by Kevin Ahern at Oregon State University on General Biochemistry. For more information about online courses go to http://ecampus.oregonstate.edu/ for the rest of the courses see http://www.youtube.com/playlist?list=PL347B70A1CC0D91C6 1. The anticodon loop has three bases complementary to the codon in the mRNA. tRNAs provide the translation function between nucleic acid sequence and amino acids. The anticodon loop frequently contains the inosine base. The base at the 3' end of the codon of the mRNA (corresponds to the base at the 5' end of the anticodon in the tRNA) is called the wobble base because it is less important for specifying the amino acid to be inserted than the first two bases. 2. Aminoacyl-tRNA synthetases have the ability to recognize and correct errors in joining of amino acids to tRNAs. For example, if one puts the wrong amino acid on the end of a tRNA and then adds an appropriate aminoacyl-tRNA synthetase, the amino acid is readily removed. 3. Two regions of aminoacyl-tRNA synthetases are important for editing - called the activation site and the editing site. 4. There are two classes of amino acid tRNA synthetases. They differ in the way they bind tRNAs and in which hydroxyl of the ribose ring they attach the amino acid to. Class I enzymes attach the amino acid to the hydroxyl on carbon #2. Class II enzymes attach the amino acid to the hydroxyl on carbon #3. 5. Base pairings in RNA are slightly different than in DNA. For example, G-U base pairs are not unstable. "I" (inosine) can also pair with C,U, or A. 6. In the genetic code, there are 64 possible combinations of the bases of the codon. Three of the possibilities (UAA, UGA, and UAG) are used as 'stop' codons. They tell the ribosomes where to stop making protein. A start codon is AUG and it codes for methionine. Since there are 61 codons used to code for amino acids and there are 20 amino acids, there is therefore 'redundancy' in the genetic code. 7. The Shine-Dalgarno sequence (GGAGG) is located near the AUG start codon in prokaryotic sequences. It is complementary to a sequence in the 16S rRNA and serves to help align the ribosome with the start site for translation in prokaryotes. 8. In prokaryotes, the first amino acid incorporated into a protein is a formylated form of methionine called fMet. The formyl group is put onto methionine after it is in the tRNA by a transformylase enzyme. Formylation of methionine in prokaryotes protects the otherwise free amino end from reacting intramolecularly and terminating transcription. 9. Peptides exit the ribosome as they are being synthesized via a tunnel in the structure. 10. Ribosomes have three sites for binding/holding/releasing tRNAs. They are called the A,P, and E sites, corresonding to the order in which tRNAs move through them (except for the very first one). 11. Initiation of protein syntheis starts with binding of IF1 and IF3 to the 30S ribosomal unit. 12. IF2 (when bound to GTP) acts to carry the Met-tRNAf to the P site of the 30S subunit and base pairs it with the AUG start codon. IF3 departs in the process. The complex of mRNA, IF1, IF2, and Met-tRNAf is called the 30S initiation complex. 13. Hydrolysis of the GTP in IF2 results in release of the IF2 and IF1 from the initiation complex. That, coupled with binding of the 50S subunit yields the 70S initiation complex with Met-tRNAf in the P site and the A and E sites open. 14. The process of elongation begins on the 70S initiation complex. EF-Tu (a G protein coupled to GTP) carries a charged tRNA to the A site of the complex. If the tRNA anti-codon base pairs properly with the codon in the mRNA, it stays matched with the codon and GTP is hydrolyzed on EF-Tu and EF-TuGDP is released. If the tRNA anti-codon does not form a stable base pairing with the complex, the entired charged tRNA-EF-Tu-GTP complex dissociates. 15. Next, the peptide group on the tRNA in the P site is transferred and covalently linked via peptide bond to the amino acid on the tRNA in the A site. This reaction is catalyzed by an enzymatic activity called peptidyltransferase - a ribozyme activity of the 23S rRNA in the 50S subunit. 16. The tRNA in the A site along with the peptide it is covalently attached to is transferred to the P stie as the "empty" tRNA in the P site is moved to the E site. EF-G-GTP is involved in the process and GTP is hydrolyzed in the process. EF-G-GTP has a similarity to the tRNAaminoacid-EF-Tu-GTP complex and may act to displace it. 17. As the old tRNA is released from the E site, the empty A site accepts the aminoacyl tRNA corresponding to the next codon. The net result of one turn of this cycle is that the polypeptide has grown by one amino acid residue and the ribosome has moved along the mRNA by three nucleotide residues. The process is repeated until a termination signal is reached.