BBMB 405 1st Edition Lecture 32 Outline of Last Lecture XV Chapter 29 RNA synthesis and Processing C Transcription in eukaryotes is highly regulated con t D The discovery of catalytic RNA was revealing in regard to both mechanism and evolution Outline of Current 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 Current Lecture XVI Chapter 30 Protein Synthesis A Protein synthesis requires translation of nucleotide sequences into amino acid sequences 1 The Ribosome a Approx 20 000 per E coli cell b 2 5 MDa 60 RNA c 3 rRNAs 52 proteins d Peptidyltransferase e 23S rRNA is catalytic 2 Stages of protein synthesis a Initiation recruitment of initiator protein 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 b Elongation use elongation factor to identify correct tRNA and to translocate use GTP hydrolysis c Termination ribosome recycled 3 Chemistry of protein synthesis peptidyl transfer a b First tRNA is a charged tRNA acetylated with correct amino acid c Forms a peptide bond 4 Protein synthesis level of accuracy a b Observed error rate is 10 4 c Evalutate probability of error for size of protein error decreases as size increases d There is a decrease in fidelity as move through central dogma e Error could take place because previous steps contained error 5 Discovery of Genetic code a Nirenburg and Matthaei discovered UUU codes for Phe b Khorana contructed own DNA and figured out codons from reconstituted Central Dogma c Holley figured out structure of tRNA 6 tRNA adapter molecules that link RNA to protein sequence a Secondary and tertiary structure are found to be conserved in all forms of life b tRNA can be edited c Inosine cause base pair change d Degeneracy arise from 3rd position e Amino acylatedtRNA is substrate of ribosome f tRNA structure is universal 7 What is the purpose of modified tRNA nucleotides a tRNAs are most heavily modified RNAs and are modified in all organisms b Structural prevent promote base pairing hydrophobic interactions with synthetases or r proteins c Alteration of codon recognition wobble position d Regulatory role transport quality control 8 tRNA CCA 3 OH end is aminoacylated 9 Wobble position reading degenerate codons a 3rd position needs to be able to be read my multiple codons b The use of inosine allow to recognize several different base pairs c d 10 Ribosomal interactions with codon anticodon a 16S rRNA contains 3 universally conserved residues b Proofread codon anticodon interaction in A site but not for wobble position c Fidelity mechanism interactions between minor grove needed for proof reading use codon and anticodon interaction this allows for non Watson Crick base pairing in the wobble position like DNA polymerase minor groove measurement of base pairs B Aminoacyl transfer RNA synthetases read genetic code 1 tRNA charging by aminoacyltRNAsynthetase where genetic code is deciphered a tRNAs must be charged with correct amino acid AminoacyltRNAsynthetases must also read genetic code Ribosome cannot determine whether a tRNA is charged with correct AA b Amino acid esters are activated intermediates on pathway to peptide bond formation Carboxyl group of amino acid attached to 2 OH or 3 OH at 3 end of tRNA 2 Amino acids are first activated by adenylation 1 reversible oxygen connecting aminoacyl AMP is from amino acid 3 Aminoacyl transfer to tRNA connect to CCA tail oxygen connecting aminoacyl tRNA from tRNA or CCA tail 4 End result a b Removing the two phosphate keeps the reaction from moving backwards pyrophsophatase removes PP c Dependent on hydrolysis of ATP d ATP that is used provides energy for protein synthesis 5 Energetics of tRNA charging a First reaction is unfavorable b Overall reaction is isoenergetic and irreversible when pyrophosphatase removes PPi c Aminoacyl tRNA hydrolysis delta G standard 29 kJ mol high transfer potential d Two high energy phosphate bonds expended for each activated amino acid 6 Pyrophosphatase drives many reactions forward and makes them irreversible 7 Aminoacyl tRNAsynthetase specificity Three similar amino acids that can be distinguished by their corresponding aminoacyltRNAsynthetases 8 Threonyl tRNAsynthetase active site a Zinc ion is coordinated with hydroxyl group so can distinguish between valine and threonine but not serine b 9 Some aminoacyl tRNAsynthetases can edit mistakes editing site is where CCA tail moves out of activation site in into editing site so it can remove wrong amino acid 10 How do aminoacyl tRNAsynthetases recognize their substrates a Multiple sites in RNA are recognized b Ensures bound to correct amino acid and codon 11 Two classes of aminoacyl tRNAsynthetase a Class I enzymes acylate the 2 OH at the 3 end of tRNA b Class II enzymes except the enzyme for Phe tRNA acylate the 3 OH c The two classes bind ATP in different conformations d Most Class I enzymes are monomeric most Class II enzymes are dimeric
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