Nucleic Acids Translation tRNA and Protein Biosynthesis

Nucleic Acids A. Nucleotides B. Nucleic Acids C. The 4 S’s 1. Size 2. Solubility 3. Shape a. A-DNA b. Z-DNA c. Topology i. Packaging ii. Supercoiling iii. Topoisomerases 4. Stability a. Nucleotides i. Tautomers ii. Acid/base b. Nucleic Acids i. Chemistry ii. Denaturation iii. Stability iv. Nucleases A. B. C. D. E. F. X4. X 5. Expression X a. Special vectors b. Fusion proteins X i. purification X ii. labeling X6. Site-directed mutagenesis Replication 1. Polymerases 2. Fidelity a.Polymerase recognition b.Exonuclease i. 3’à5’ ii. 5’à3’ c.Mis-match repair d.Post-replication repair i. Direct reversal ii. Base excision iii. Nucleotide excision 3. Sequence determination 4. PCR G. Transcription D. Structure of the Information 1. Exceptions to flow 2. Structure 3. Levels of Control E. A. X B. X 1. 2. 3. 4. Overview-mRNA structure Process RNA Polymerase Fidelity C. X Recombinant DNA: Biochemical D. X Basis of Biotechnology 1. 2. 3. 4. Restriction enzymes, DNA ligase E. X F. X DNA (rDNA) Vectors and Inserts to make recombinant G. X Transformation of hosts H.Translation Selection of transformants a. b. Selectable marker/gene Distinguish empty plasmids i. Loss of resistance ii. Reporter gene 1. Genetic Code a. triplet b. deciphering Transcription & Translation Transcription Overview Process RNA Polymerase Fidelity Translation Genetic Code triplet deciphering tRNA Structure Anticodon Acylation (charging) Aminoacyl-tRNA Synthetases Mechanism Fidelity Protein Biosynthesis Overview Process Ribosome review Peptidyl Transferase Fidelity 1 Translation: tRNA X-Ray Structure of Yeast tRNAPhe Translation: tRNA tRNA anticodon-codon interactions e.g., Met, Trp e.g., Ile, many others: Ala 2 Translation: tRNA Acylation = Charging of tRNA THE KEY ENZYMES FOR THIS ARE THE Guardians of the CODE How do they recognize the correct tRNA? Translation: tRNA Class I and II Aminoacyl-tRNA synthetases Class II Class I Minor groove; Makes 3’-end a hairpin Recognizes Anticodon loop Major groove; 3’end remains helical Doesn’t recognize Anticodon loop HO O 3 Translation: tRNA Aminoacylation: 2 step reaction: 1) A.A. + ATP à AA-AMP + PPi 2) AA-AMP + tRNA à AA-tRNA + AMP (AA-tRNA is shown at right = “charged” tRNA) 2 steps Again, just like with DNA and RNA synthesis, subsequent hydrolysis of pyrophosphate provides driving force for reaction First ½ reaction is sequential random bi uni AA-tRNA A.A. ATP AMP E•AMP-A.A. E•ATP•A.A. E tRNA PPi E E•AMP-A.A.•PPi ‡ ATP * A.A. Translation: tRNA Aminoacylation: 2 step reaction: 1) A.A. + ATP à AA-AMP + PPi 2) AA-AMP + tRNA à AA-tRNA + AMP (AA-tRNA is shown at right = “charged” tRNA) 2 steps Again, just like with DNA and RNA synthesis, subsequent hydrolysis of pyrophosphate provides driving force for reaction First ½ reaction is sequential random bi uni AA-tRNA A.A. ATP E•AMP-A.A. E•ATP•A.A. E tRNA PPi E•AMP-A.A.•PPi ‡ ATP AMP E * A.A. 4 Translation: tRNA E•AMP-Tyr This bi-pyrimidal shape has 180° angle between axial oxygens, which were 109.5° in ATP. [E•AMP∼Tyr∼PPi]‡ ‡ Condensation of two acids = anhydride (mixed) Translation: tRNA Class II How do they ensure the correct amino acid is attached to the correct tRNA? Class I - tRNAIle Some amino acids are different enough, e.g., Phe versus Tyr (H-bonds from D176 & Y34 to phenolic oxygen account for 150,000 fold stronger binding) 5 Translation: tRNA The extra methylene only gets Eile 200-fold stronger binding to Ile versus Val. And [Val] is 5x higher than [Ile]. This cuts the effective driving force to only 40-fold = 2.5% error rate. The editing site comes to the rescue! When tRNAile binds, the Val-AMP is forced into the hydrolysis site. Ile-AMP is too large to fit. Decreases the error rate to 0.0003. Too small for Ile Incorrect Correct Translation: tRNA Hydroxyl group where m ethyl val Threonine val Hydrolytic Site: Too polar for Val valine threonine Acylation Site Hydrolysis Correct Incorrect val 6 Transcription & Translation Transcription Overview Process RNA Polymerase Fidelity Translation Genetic Code triplet decyphering tRNA Structure Anticodon Acylation (charging) Aminoacyl-tRNA Synthetases Mechanism Fidelity Protein Biosynthesis Overview Process Ribosome review Peptidyl Transferase Fidelity Translation: Protein Biosynthesis Making the Peptide Bond 7 Translation: Protein Biosynthesis Process: •Initiation Fidelity •Elongation 1. Decoding 2. Peptidyl Transferase 3. Translocation •Termination Making the Peptide Bond Translation: Protein Biosynthesis 8 Translation: Protein Biosynthesis Ribosome: RNA-protein complex Translation: Protein Biosynthesis Process Overview: • mRNA read in 5’à3’ direction • protein synthesized from N-terminus to C-terminus • protein chain elongation occurs by transferring new AA to C-terminus of growing chain Ribosome has 3 tRNA binding sites: A-site – amino acyl tRNA binding site P-site – peptidyl-tRNA binding site E-site – exit site, deacylated tRNA tide pep Poly nel Tun 9 Translation: Protein Biosynthesis Process Overview: • mRNA read in 5’à3’ direction • protein synthesized from N-terminus to C-terminus • protein chain elongation occurs by transferring new AA to C-terminus of growing chain Ribosome has 3 tRNA binding sites: A-site – amino acyl tRNA binding site P-site – peptidyl-tRNA binding site E-site – exit site, deacylated tRNA nel Tun tide pep Poly Translation: Protein Biosynthesis Translation Initiation Sequences Consensus Shine-Delgarno mRNA sequence 5’-AAGGAGGU-3’ 10 Translation: Protein Biosynthesis Elongation Recall: 1 2 EF-Tu Decoding Decoding (GTP (GTP hydrolysis) hydrolysis) GTP Peptidyltransferase Peptidyltransferase 3 Translation: Protein Biosynthesis Elongation 11 Translation: Protein Biosynthesis Elongation: Decoding EF-Tu: • Most abundant protein • Binds to AA-tRNA (not free tRNA) • Anti-codon end is free to bind to 30S-mRNA. Decoding Kinetic proofreading: GTP hydrolysis P A O H E E P/E A/P Translation: Protein Biosynthesis Elongation: Decoding Bound A-site Empty A-site 5’-UUU 3’-AAG Three highly conserved residues on the 30S subunit each associate with the tRNA anticodon residues: • Position 1 (U1): A1493 • Position 2 (U2): A1492 • Position 3 (Wobble) (U3): G530 Wobble Position 2 Position 1 Minor groove mRNA tRNA rRNA (30S) rProtein S12 EF-Tu These highly conserved residues all interact with the minor groove!! Where have we seen this before? The energetics of these binding interactions is insufficient to account for the error rate of protein synthesis…. 12 Translation: Protein Biosynthesis Elongation: Fidelity How does the EF-Tu•GTP•AA-tRNA•mRNA•Ribosome Complex examine this codon-anticodon interaction via a second method? (much like DNA polymerases and aminoacyl-tRNA synthetases) It uses a complex of EF-Tu•GDP•AA-tRNA•mRNA•Ribosome to test the codonanticodon interaction via a conformational change that stresses this interaction. • EF-Tu•GTP•AA-tRNA binds the A-site with a strained anticodon stem-loop • Anticodon-codon interactions in the A-site induce EF-Tu’s hydrolysis of GTP to GDP. GTP hydrolysis is FASTER for cognate tRNA. • This results in EF-Tu release from the complex as EF-Tu•GDP • Once the EF-Tu is gone, the AA-tRNA relaxes, swings its acceptor stem into the A-site on the 50S, pivoting at the codon-anticodon interaction • Non-cognate tRNAs do not survive this pivot and fall out most of the time THEREFORE, GTP HYDROLYSIS IS KEY: 1. In its slowness, time is allowed for optimal cognate codon-anticodon interactions 2. In its hydrolysis, initiated by these interactions, the process achieves a second test of the fitness by being ”hinged” at the codon-anticodon during the pivoting. Translation: Protein Biosynthesis Frequency of inserting an incorrect amino acid p = (1 – e)n p is the probability of an error-free protein e is the error rate n is the length of the protein Each of the 2 selection mechanisms (EF-Tu GTPase and conf. change) have a 1% error rate (e = 10-2). Combined e = 10-4. 13 Translation: Protein Biosynthesis Elongation: Transpeptidation P A O H E E Transpeptidation P/E A/P Translation: Protein Biosynthesis Elongation: Transpeptidation or the Peptidyltransferase reaction Peptidyl-tRNA (in the P-site) Gly Polarization of a-amino of incoming amino acid to make it a better nucleophile is from the N3 of Adenine2486 of the large rRNA. It’s a RIBOZYME!! Rate enhancement is mostly all proximity (entropy trap) Ala Aminoacyl-tRNATyr rRNA (in the A-site) 14 mRNA Interactions with tRNAs P A E mRNA 5’ 3’ Translation: Protein Biosynthesis Elongation: Decoding What happens to EF-Tu•GDP? P A O H E E P/E A/P 15 Translation: Protein Biosynthesis Elongation: Decoding EF-Ts is a GTP exchange protein (guanine nucleotide exchange factor; GEF) EF-Tu•GDP Ts Ts Ts Translation: Protein Biosynthesis ENERGY REQUIREMENTS: • 2 ATP equivalents for every base in the 3-base codon of the mRNA =6 (mRNA synthesis (transcription)) • 2 ATP equivalents for every AA-tRNA =2 (tRNA charging) • 1 ATP equivalent for binding with EF-Tu • 1 ATP equivalent for translocation with EF-G =1 =1 = 10 total ATPs for every residue 16 END of Material for Exam 4 17