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Chapter 14 Translation 18 and 20 October, 2004 Overview • Translation uses the nucleotide sequence of mRNA to specify protein sequence. • Each ORF specifies a polypeptide. • Ribosome components and / or tRNAs recognize structures and sequences near the 5’ end of the transcript to identify the correct start codon. • tRNAs are highly modified short RNAs that are the adaptors between codons and amino acids. • Amino acyl tRNA synthetases recognize structural features of tRNAs and charge only the correct tRNA with the correct amino acid. • The large and small ribosomal subunits are extremely complex ribonucleoprotein structures that dissociate and reassociate in each round of translation. • Peptide synthesis is catalyzed by a ribozyme, and proceeds in the N-to-C terminal direction. • The ribosome uses three tRNA binding sites: A, P, and E. • tRNAs are delivered to the ribosome by EF-Tu. • EF-G GTP hydrolysis along with peptide bond formation drive ribosomal translocation. • Translation termination involves release factors and GTP hydrolysis. • Translation-dependent RNA stability assures the degradation of damaged messages. Three possible open reading frames. Shine-Dalgarno and Kozak Sequences Kozak: Identification of Consensus Kozak: Correct context makes a better barrier to downstream initiation. tRNA Structures Two-step charging of tRNA Two-step charging of tRNA tRNA Structural Elements Recognized by Aminoacyl-tRNA Synthetase Synthetase-tRNA cocrystal The Problem Solved by Editing Pockets The ribosome cannot distinguish incorrectly charged tRNAs There are twenty-one amino acids. Prokaryotic transcription and translation are linked. Composition of Ribosomes Translation Overview The Peptidyl Transferase Reaction The Ribosome Ribosome - tRNA interactions tRNA Interactions Within the Ribosome Ribosome Channels Initiation in Prokaryotes Initiation in Prokaryotes Initiation in Prokaryotes Initiation in Eukaryotes Start Codon Identification Interactions between PABP and eIF4F circularize the transcript. uORFs IRES Aminoacyl- tRNAs bind to the ribosome in a complex with EF-Tu. Ef-Tu release requires correct base pairing. The ribosome also uses minor-groove interactions between the 16S rRNA and the codon-anticodon to drive correct base pairing Accommodation (rotation) of the tRNA strains the codon-anticodon interaction causing incorrectly paired tRNAs to dissociate. Peptidyl Transferase Ribozyme Peptide bond formation and EF-G GTP hydrolysis drive translocation. EF-G is a structural homolog of EF-Tu-tRNA GTP hydrolysis drives conformational change. Peptide anticodons allow release factors to recognize the stop codon. GGQ on the RF-I stimulates peptidyl transfer to water. RRF and EF- G stimulate dissociation of the terminated ribosome. tmRNA and SsrA rescue stalled complexes Normal translation displaces exon-junction complexes. Nonsense-mediated decay is caused by undisplaced exon-junction complexes. In eukaryotes, abnormal termination causes message degradation. Title Title
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