16 The roles of RNA in protein synthesis Copyright (c) by W. H. Freeman and Company
17 The three roles of RNA in protein synthesis Three types of RNA molecules perform different but complementary roles in protein synthesis (translation)Messenger RNA (mRNA) carries information copied from DNA in the form of a series of three base “words” termed codonsTransfer RNA (tRNA) deciphers the code and delivers the specified amino acidRibosomal RNA (rRNA) associates with a set of proteins to form ribosomes, structures that function as protein-synthesizing machinesCopyright (c) by W. H. Freeman and Company
18 The folded structure of tRNA specifies its decoding function Figure 4-26Copyright (c) by W. H. Freeman and Company
19 Aminoacyl-tRNA synthetases activate amino acids by linking them to tRNAs Each tRNA molecule is recognized by a specific aminoacyl-tRNA synthetase
20 Fidelity of protein synthesis determined by: Correct aminoacylation of tRNACodon-anticodon pairing
21 Double sieve mechanism for error correction Aminoacyl tRNA synthetases -at least one for every amino acid -for different codons have different synthetases -error correction lies in specificity of synthetase and tRNA. No mechanism exists for error correction once tRNA is mischarged and separated from synthetaseDouble sieve mechanism for error correctionSynthetases have 2 sites: active site, hydrolytic site.Amino acids larger than the correct amino acid are never activated because they are too large to fit into the active site. Smaller amino acids (than the correct one) fit into the hydrolytic site (which excludes the correct amino acid) and are hydrolyzed.
22 Nonstandard base pairing often occurs between codons and anticodons
26 Initiation mRNA binds to ribosome Selection of initiation codon Binding of charged initiator tRNA (first amino acid)
27 Initiation Formation of 30S preinitiation complex 30 S subunit (contains 16S rRNA), mRNA, charged tRNA f-met, initiation factors, GTP+ 50S subunit (GTP hydrolysis)Resulting in formation of the 70S initiation complexfmet-tRNA is fixed into the “P site”reading frame is now determined.
31 Initiation of eukaryotic protein synthesis generally occurs at the 5’ end of mRNA but may occasionally occur at internal sites
32 Initiation of prokaryotic protein synthesis generally occurs at the Shine Delgarno site The untranslated leader or 5’ end of prokaryotic mRNAs contain a ribosome binding site (rbs) or Shine Delgarno site located upstream of the AUG and complementary to the 3’ end of the 16S rRNA.mRNA: 5’ ….AGGAGGU……………..AUG3’end of 16S rRNA 3’ ...UCCUCCA……………………..I I I I I I I
35 Elongation Peptide bond formation Movement of mRNA/ ribosome (translocation) so each codon may be “read”
36 Elongation Requirements: Elongation factors and GTP hydrolysis Occupation of “A” site by next tRNAPeptide bond formed by peptidyl transferase enzymeUncharged tRNA-fmet in P site and dipeptidyl tRNA in A siteTranslocation:deacylated tRNA fmet leaves P sitepeptidyl tRNA moves from A to P sitemRNA moves 3 bases to position next codon at A site
42 when termination codons are reached (UGA, UAA, UAG) Completed protein is dissociated from machineryRibosome released
43 when termination codons are reached (UGA, UAA, UAG) Peptidyl tRNA moves from A to P siteRelease factors (RF) recognize specific stop codonsRF forms activated complex with GTPActivated complex binds to termination codon and alters specificity of peptidyl transferaseIn presence of RF, peptidyl transferase catalyzes reaction of bound peptidyl moiety with water instead of with free aminoacyl tRNARelease of polypeptideDissociation of 70S ribosome into 50S and 30S subunits.
44 Summary of Protein Synthesis 1. Binding of mRNA to ribosome2. Charged, amino-acylated initiator tRNA binds to P site of ribosome and is based paired through tRNA anticodon to codon on mRNA3. A second amino-acylated tRNA fills A site and anticodon H-bonds with second codon on mRNA4. Amino acids in P and A site are joined by a peptide bond.tRNA in P site is released.tRNA (with 2 amino acids joined) in A site moves to P siteA new amino-acylated tRNA moves into A site by anticodon-codon pairing5. Step (4) is repeated until codon in A site is a stop codon; peptide is released.
45 Post-translational Modifications (Bacteria) removal of formyl groups (fmet)removal of first few amino acids (aminopeptidase)glycosylation (affects targeting, activity)phosphorylation (by kinases)S-S bond formationPolypeptide cleavage-removal of transit peptide upon organelle import-removal of signal sequence (ER secretion)-activation of enzymes