BBMB 405 1st Edition Lecture 33 Outline of Last Lecture XVI Chapter 30 Protein Synthesis A Protein synthesis requires translation of nucleotide sequences into amino acid sequences B Aminoacyl transfer RNA synthetases read genetic code Outline of Current Lecture XVI Chapter 30 Protein Synthesis C The ribosome is the site of protein synthesis Current Lecture XVI Chapter 30 Protein Synthesis C The ribosome is the site of protein synthesis 1 The ribosome at atomic resolution Use cryo EM prove ribosome make of RNA and RNA is catalytic component of ribosome 2 Composition of bacterial ribosome a Complex of secondary tertiary structures and proteins b Small Subunit 30S has 16S rRNA and Large subunit 50S has 5S and 23S rRNA c Use base pair structure to predict secondary structure Sequence ribosomal RNA from different genomes and see overall structure Same sequence same structure d Secondary structure from evolutionary stand point Comparison of prokaryotic and eukaryotic ribosomes Conserved through all domains of life Ribosome build on each other as organism gets more complex 3 tRNAs bind in A site then proceed through P and E sites a A site Aminoacyl site P site Peptidyl site E site Exit site b The P site has tRNA with nascent peptide c As mRNA passes through channel only A and P site have tRNA attached to it 4 30S contains mRNA binding cleft 50S contains peptidyltransferase active site These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute 5 Ribosome initiation in prokaryotes a Initiation in prokaryotes is purine rich Shine Dalgarno sequence pairs with 16S rRNA b The 3 end of 16S rRNA pairs with the Shine Dalgarno sequence if mutation in the 16S rRNA it would disrupt the pairing of it with the SD sequence and would cause defects in translation initiation and result in growth defects 6 The initiator tRNA is acylated with N formylmethionine fMet a Only one Met codon AUG b Two different tRNAs carry Met tRNAf Reads start codon tRNAm Reads internal Met positions c Only one aminoacyl tRNAsynthetase for Met addition d Formyl group is added specifically to Met tRNAf e Machinery for translation only recognizes formylated Met f 7 Translation initiation requires initiation factors Prokaryotic initiation a Initaition factors 1 and 3 IF1 and IF3 prevent premature 50S subunit joining need to make sure everything is in proper place before 50S binds fidelity of initation b Initationn factor 2 IF2 recruits fMet tRNAf to the P site of initation complex c Release of IF1 and IF3 is necessary for 50S subunit joining d 50S joining is accompanied by GTP hydrolysis by IF2 and IF2 release e 8 Charged tRNAs are delivered to A site by elongation factor Tu EF Tu a EF Tu protects aa tRNA ester bond b GTP hydrolysis when proper codon is present proofreading 9 Peptide bond formation at peptidyltransferase center a b Thermodynamically spontaneous reaction Spontaneous if interact correctly No acid base chemistry going on so pH independent pKa of nucleotides are not contributing c Catalysis by proximity and orientation Positioning of substrates helps lower activation entropy Electrostatic forces in molecule stabilizes intermediates in reaction 10 Translation elongation 11 Multiple ribosomes translate mRNAs at once coordination of transcription and translation everything is processed 5 to 3 12 Translation termination a Release factors are required for termination b RF1 and RF2 recognize stop codons not tRNA and promote water molecule attack on ester linkage leading to peptide release c Ribosome recycling promoted by EF G and ribosome release factor use GTP hydrolysis d D Eukaryotic Translation 1 Main differences from prokaryote initiation a No Shine Dalgarno but use 5 cap for recognition b Not cotranscriptional c Initiating Met is not formylated but there is a specialized initiator tRNA MettRNAi d Scanning to find start codon is ATP dependent e Eukaryotes can recognize fMet tRNA of bacteria and use this in immune defense f
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