BBMB 405 1st Edition Lecture 32Outline of Last Lecture XV. Chapter 29: RNA synthesis and ProcessingC. Transcription in eukaryotes is highly regulated (con’t)D. The discovery of catalytic RNA was revealing in regard to both mechanism and evolutionOutline of Current Lecture XVI. Chapter 30: Protein SynthesisA. Protein synthesis requires translation of nucleotide sequences into amino acid sequencesB. Aminoacyl-transfer RNA synthetases read genetic codeCurrent LectureXVI. Chapter 30: Protein SynthesisA. Protein synthesis requires translation of nucleotide sequences into amino acid sequences1. The Ribosomea. Approx. 20,000 per E. coli cellb. 2.5 MDa, 60% RNAc. 3 rRNAs, 52 proteinsd. Peptidyltransferasee. 23S rRNA is catalytic2. Stages of protein synthesisa. Initiation: recruitment of initiator proteinThese 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 hydrolysisc. Termination: ribosome recycled3. Chemistry of protein synthesis: peptidyl transfera.b. First tRNA is a charged tRNA (acetylated with correct amino acid)c. Forms a peptide bond4. Protein synthesis level of accuracya.b. Observed error rate is 10^-4c. Evalutate probability of error for size of protein, error decreases as size increasesd. There is a decrease in fidelity as move through central dogmae. Error could take place because previous steps contained error5. Discovery of Genetic codea. Nirenburg and Matthaei: discovered UUU codes for Pheb. Khorana: contructed own DNA and figured out codons from reconstituted Central Dogmac. Holley: figured out structure of tRNA6. tRNA adapter molecules that link RNA to protein sequencea. Secondary and tertiary structure are found to be conserved in all forms of lifeb. tRNA can be editedc. Inosine: cause base pair changed. Degeneracy arise from 3rd positione. Amino-acylatedtRNA is substrate of ribosomef. tRNA structure is universal7. What is the purpose of modified tRNA nucleotides?a. tRNAs are most heavily modified RNAs, and are modified in all organismsb. Structural: prevent/promote base pairing, hydrophobic interactions with synthetases or r-proteinsc. Alteration of codon recognition: wobble positiond. Regulatory role: transport, quality control8. tRNA CCA-3’-OH end is aminoacylated9. Wobble position: reading degenerate codonsa. 3rd position needs to be able to be read my multiple codonsb. The use of inosine: allow to recognize several different base pairsc.d.10. Ribosomal interactions with codon-anticodona. 16S rRNA contains 3 universally conserved residuesb. Proofread codon-anticodon interaction in A-site but not for wobble positionc. 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 code1. tRNA charging by aminoacyltRNAsynthetase – where genetic code is deciphereda. tRNAs must be charged with correct amino acid- AminoacyltRNAsynthetases must also read genetic code- Ribosome cannot determine whether a tRNA is charged with correct AAb. 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 tRNA2. Amino acids are first activated by adenylation (1): reversible, oxygen connecting aminoacyl-AMP is from amino acid3. Aminoacyl transfer to tRNA: connect to CCA tail; oxygen connecting aminoacyl-tRNA from tRNA or CCA tail4. End result:a.b. Removing the two phosphate keeps the reaction from moving backwards; pyrophsophatase removes PPc. Dependent on hydrolysis of ATPd. ATP that is used provides energy for protein synthesis5. Energetics of tRNA charginga. First reaction is unfavorableb. Overall reaction is isoenergetic and irreversible when pyrophosphatase removes PPic. Aminoacyl-tRNA hydrolysis (delta)G’(standard) = -29 kJ/mol high-transfer potentiald. Two high-energy phosphate bonds expended for each activated amino acid6. Pyrophosphatase drives many reactions forward and makes them irreversible7. Aminoacyl-tRNAsynthetase specificity: Three similar amino acids that can be distinguished by their corresponding aminoacyltRNAsynthetases8. Threonyl-tRNAsynthetase active sitea. Zinc ion is coordinated with hydroxyl group so can distinguish between valine and threonine but not serineb.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 acid10. How do aminoacyl-tRNAsynthetases recognize their substratesa. Multiple sites in RNA are recognizedb. Ensures bound to correct amino acid and codon11. Two classes of aminoacyl-tRNAsynthetasea. Class I enzymes acylate the 2’-OH at the 3’ end of tRNAb. Class II enzymes (except the enzyme for Phe-tRNA) acylate the 3’-OHc. The two classes bind ATP in different conformationsd. Most Class I enzymes are monomeric, most Class II enzymes are