Transcription & Translation, Exams of Topology

Download Transcription & Translation and more Exams Topology in PDF only on Docsity! 11/15/21 1 Lecture 25 (11/15/21) 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.Translation • Reading: Ch26; 960-964 Ch27; 1006- 1013, 1018- 1024 • Problems: Ch26; 1,2,6, 11,17 Ch27; 5,8,9,11 NEXT • Reading: Ch27; 1015- 1018, 1028- 1036 • Problems: Ch27; 6,7,10, 12,13,15, 16,18,19 Transcription & Translation Transcription Overview-mRNA structure 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 11/15/21 2 Nucleic acid function: Central Dogma Transcription mRNA structure 5’ cap and 3’ polyA tail 5’ prokaryotes eukaryotes Start of Translation End of Translation AUG OPEN READING FRAME (ORF)5’-UTR 3’-UTR Shine-Delgarno 5’-cap 3’-Poly(A) Stop Codon Transcription AAGGAGGU 3’ …AAAAAAAAAA poly(A) polymerase (PAP) poly(A)-binding protein (PABP II) 11/15/21 5 RNA Polymerase Structure RNA pol is a multi-subunit enzyme These subunits make up the core complex: • two α subunits make non-specific contacts with DNA for positioning • the β and β’ subunits catalyze the addition of ribonucleotides to the growing chain • the ω subunit acts to stabilize the complex (a2bb’ws) Transcription Nascent RNA Subunit Gene MW # Role a2 rpoA 34 2 Non-specific DNA binding b rpoB 150 1 Polymerase b’ rpoC 155 1 Non-specific DNA binding & polymerase w rpoZ 10 1 Zn2+ binding s rpoD 70 1 Promoter recognition Template DNA Coding strand DNA RNA•DNA duplex is in the A-form It uses the same mechanism for correct W-C bp and fidelity Therefore, error rate is the same 1/10,000 Rate is ~ 50 base/sec Processivity is ~2000 bp No 3’à5’ exonuclease proofreading Coding strand Template strand Yeast RNA polymerase PDBid 1I50 Transcription Eukaryotic RNA Polymerase II Conformations Closed conformation Also, the b’ homolog has a disordered CTD that is phosphorylated In going from Initiation to elongation Open conformation 11/15/21 6 P-site Funnel Pore RNA polymerase II PDBid 2E2H Transcription Eukaryotic RNA Polymerase II NTP sites (A & E) i + 1 Bridge E-site A-site RNAP is very sensitive to the helix shape in the active site. If there is a mispaired nucleotide, or even a deoxy nucleotide, it stalls (as well as at damaged DNA). The helix unwinds and the 3’-end of the RNA goes into a P-site. Other subunits/proteins hydrolyze RNA at 3’-end. i –1 Backtracking Proofreading 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 11/15/21 7 Nucleic acid function: Central Dogma Transcription & Translation Recall: Genetic Code is Degenerate & Nonrandom Gold = hydrophobic amino acids; pyrimidine at second position Polar amino acids (blue = basic; red = acidic; purple = uncharged polar) have purine at second position Translation: The Genetic Code How was the Triplet code discovered?