Transcription & Translation, Lecture notes of Topology

Download Transcription & Translation and more Lecture notes Topology in PDF only on Docsity! 11/17/21 1 Lecture 26 (11/17/21) 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 D. Structure of the Information 1. Exceptions to flow 2. Structure 3. Levels of Control E. Recombinant DNA: Biochemical Basis of Biotechnology 1. Restriction enzymes, DNA ligase 2. Vectors and Inserts to make recombinant DNA (rDNA) 3. Transformation of hosts 4. Selection of transformants a. Selectable marker/gene b. Distinguish empty plasmids i. Loss of resistance ii. Reporter gene 4. X 5. Expression a. Special vectors b. Fusion proteins i. purification ii. labeling 6. Site-directed mutagenesis A. X B. X C. X D. X E. X F. 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 1. Overview-mRNA structure 2. Process 3. RNA Polymerase 4. Fidelity A. X B. X C. X D. X E. X F. X G. X H.Translation 1. Genetic Code a. triplet b. deciphering • Reading: Ch27; 1015- 1018, 1028- 1036 • Problems: Ch27; 6,7,10, 12,13,15, 16,18,19 NEXT (after Thanksgiving; will post) 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 11/17/21 2 Translation: tRNA X-Ray Structure of Yeast tRNAPhe tRNA anticodon-codon interactions Translation: tRNA e.g., Ile, many others: Ala e.g., Met, Trp 11/17/21 5 ‡ Translation: tRNA [E•AMP∼Tyr∼PPi]‡ E•AMP-Tyr Condensation of two acids = anhydride (mixed) This bi-pyrimidal shape has 180° angle between axial oxygens, which were 109.5° in ATP. How do they ensure the correct amino acid is attached to the correct tRNA? Translation: tRNA 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) Class II Class I - tRNAIle 11/17/21 6 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. Correct Incorrect Too small for Ile threonine Correct Incorrect Translation: tRNA valine val val val Threonine Hydroxyl group where methyl Acylation Site Hydrolytic Site: Too polar for Val Hydrolysis 11/17/21 7 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 11/17/21 10 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 Translation: Protein Biosynthesis Polypeptide Tunnel Translation Initiation Sequences Translation: Protein Biosynthesis 5’-AAGGAGGU-3’Consensus Shine-Delgarno mRNA sequence 11/17/21 11 Elongation Translation: Protein Biosynthesis Decoding (GTP hydrolysis) Peptidyltransferase GTP EF-Tu Decoding (GTP hydrolysis) Peptidyltransferase 1 2 3 Recall: video of translation: http://www.hhmi.org/biointeractive/translation-advanced- detail Elongation Translation: Protein Biosynthesis 11/17/21 12 Translation: Protein Biosynthesis Kinetic proofreading: GTP hydrolysis OH E P A E P/E A/P Decoding Elongation: Decoding EF-Tu: •Most abundant protein •Binds to AA-tRNA (not free tRNA) •Anti-codon end is free to bind to 30S-mRNA. Translation: Protein Biosynthesis Elongation: Decoding 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 Position 1 Position 2 Wobble These highly conserved residues all interact with the minor groove!! Where have we seen this before?Minor groove The energetics of these binding interactions is insufficient to account for the error rate of protein synthesis…. Empty A-site Bound A-site mRNA tRNA rRNA (30S) rProtein S12 EF-Tu 5’-UUU 3’-AAG 11/17/21 15 mRNA Interactions with tRNAs Interactions of tRNA with mRNA PDBid 2WDK AP E mRNA 5’ 3’ Translation: Protein Biosynthesis What happens to EF-Tu•GDP? OH E P A E P/E A/P Elongation: Decoding 11/17/21 16 Translation: Protein Biosynthesis Elongation: Decoding Ts Ts Ts EF-Ts is a GTP exchange protein (guanine nucleotide exchange factor; GEF) EF-Tu•GDP Translation: Protein Biosynthesis ENERGY REQUIREMENTS: •2 ATP equivalents for every base in the 3-base codon of the mRNA (mRNA synthesis (transcription)) •2 ATP equivalents for every AA-tRNA (tRNA charging) •1 ATP equivalent for binding with EF-Tu •1 ATP equivalent for translocation with EF-G = 6 = 2 = 1 = 1 = 10 total ATPs for every residue 11/17/21 17 END of Material for Exam 4