BBMB 405 1st Edition Lecture 33Outline of Last Lecture XVI. Chapter 30: Protein SynthesisA. Protein synthesis requires translation of nucleotide sequences into amino acid sequencesB. Aminoacyl-transfer RNA synthetases read genetic codeOutline of Current Lecture XVI. Chapter 30: Protein SynthesisC. The ribosome is the site of protein synthesisCurrent LectureXVI. Chapter 30: Protein SynthesisC. The ribosome is the site of protein synthesis1. The ribosome at atomic resolution: Use cryo EM, prove ribosome make of RNA and RNA is catalytic component of ribosome2. Composition of bacterial ribosomea. Complex of secondary, tertiary structures, and proteinsb. Small Subunit (30S) has 16S rRNA and Large subunit (50S) has 5S and 23S rRNAc. Use base pair structure to predict secondary structure- Sequence ribosomal RNA from different genomes and see overall structure- Same sequence same structured. 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 complex3. tRNAs bind in A site, then proceed through P and E sitesa. A site = Aminoacyl site; P site = Peptidyl site; E site = Exit siteb. The P site has tRNA with nascent peptidec. As mRNA passes through channel only A and P site have tRNA attached to it4. 30S contains mRNA binding cleft, 50S contains peptidyltransferase active siteThese 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 prokaryotesa. Initiation in prokaryotes is purine rich Shine-Dalgarno sequence, pairs with 16S rRNAb. The 3’ end of 16S rRNA pairs with the Shine-Dalgarno sequence; if mutation in the16S rRNA it would disrupt the pairing of it with the SD sequence and would cause defects in translation initiation and result in growth defects6. 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 internalMet positionsc. Only one aminoacyl-tRNAsynthetase for Met additiond. Formyl group is added specifically to Met-tRNAfe. Machinery for translation only recognizes formylated Met f.7. Translation initiation requires initiation factors; Prokaryotic initiationa. 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 initationb. Initationn factor 2 (IF2) recruits fMet-tRNAf to the P site of initation complexc. Release of IF1 and IF3 is necessary for 50S subunit joiningd. 50S joining is accompanied by GTP hydrolysis by IF2 and IF2 releasee.8. Charged-tRNAs are delivered to A-site by elongation factor Tu (EF-Tu)a. EF-Tu protects aa-tRNA ester bondb. GTP hydrolysis when proper codon is present, proofreading9. Peptide bond formation at peptidyltransferase centera.b. Thermodynamically spontaneous reaction- Spontaneous if interact correctly- No acid-base chemistry going on so pH independent- pKa of nucleotides are not contributingc. Catalysis by proximity and orientation- Positioning of substrates helps lower activation entropy- Electrostatic forces in molecule stabilizes intermediates in reaction10. Translation elongation11. Multiple ribosomes translate mRNAs at once: coordination of transcription and translation, everything is processed 5’ to 3’12. Translation terminationa. Release factors are required for terminationb. RF1 and RF2 recognize stop codons (not tRNA) and promote water molecule attack on ester linkage leading to peptide releasec. Ribosome recycling promoted by EF-G and ribosome release factor: use GTP hydrolysisd.D. Eukaryotic Translation1. Main differences from prokaryote initiationa. No Shine-Dalgarno but use 5’ cap for recognitionb. Not cotranscriptionalc. Initiating Met is not formylated, but there is a specialized initiator tRNA (Met-tRNAi)d. Scanning to find start codon is ATP dependente. Eukaryotes can recognize fMet-tRNA of bacteria and use this in immune
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