BBMB 405 1nd Edition Lecture 28Outline of Last Lecture XIV. Chapter 28: DNA Replication, Repair, and RecombinationD. DNA recombination plays important roles in replication, repair, and other processesXV. Chapter 29: RNA Synthesis and ProcessingA. ReviewB. RNA Polymerases catalyze transcriptionOutline of Current Lecture XV. Chapter 29: RNA synthesis and ProcessingB. RNA polymerases catalyze transcription (con’t)Current LectureXV. Chapter 29: RNA synthesis and ProcessingB. RNA polymerases catalyze transcription1. First step of Transcription:a. RNA doesn’t need a primerb. First step is connecting annealing two NTPsc. No reaction hydrolyses 5’ end of nucleotide which protects it because triphosphate must be taken off before degraded2. Nucleotide addition and translocation by RNAPThese 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.3. DNA is unwound locally at transcription bubble4. Channels with RNA polymerasea. Different structures help with unwindingb. Separate channels bring in nucleotidesc.5. Ds DNA and DNA-RNA unwinding are coupled with translocationa. ds DNA unwinding: Phe used as wedgeb. Diphosphate still bound – release pushes reaction forwardc. If diphosphate is still bound, there is a possibility of breaking newly formed bondd. DNA-RNA unwinding: wedge allows for breaking of hybrid6. RNA polymerases can backtrack for proofreadinga. Mismatches promote backtracking because more energetically favorable to break non Watson and Crick base pairsb. There are no further proofreading mechanismsc. More error prone than DNA polymerased. Will pause where nucleotide incorporated but not move to next site, will move back and get stuck7. Bacterial Transcription Initiationa. RNA polymerases initiate transcription from specific sequences called promoter sites (or just promoters): promoters can be constitutive (always on) or regulated (activated or repressed)b. RNAP holoenzyme is required for initiation (includes sigma subunit)8. RNA polymerase binds to promoter sites on DNA template to initiate transcriptiona. Consensus sequence is the most common sequence at each positionb. Closer the sequence is to the consensus sequence the stronger and more frequently the promoter will bindc. Promoter strength regulates transcriptiond. UP elements are located before -35 region and are recognized by alpha subunite.9. Sigma subunits of RNA polymerase recognize two promoter elementsa. Different sigma factors bind different promotersb. Specific genes are expressed in response to various stresses, when different nutrients are limited different sigma subunits turn on so different promoters are turned onc. Size of gap effects affinity of sigma10. Promoter binding leads to DNA unwindinga. Closed complex: recognition of dsDNA elementsb. Open complex: -35 site is not unwound, -10 site is unwoundc. Initially transcribing complex: prone to abortive <10 nt transcriptsd. Elongation complex (sigma dissociated): escaped from promoter11. Bacterial transcription terminationa. Rho-independent: RNA polymerase, RNA synthesis, RNA structure, RNA release- GC rich palindromic sequence- Termination signals: GC rich sequences form hairpins, downstream U tracks- Hairpin formation leads to RNAP stalling with RNA template hybrid composed of rU-dA base pairs, weak base pairs RNA dissociation-b. Rho-dependent: Rho binding, rho migration, RNA release12. Metabolite sensing RNA structures called “riboswitches” can control terminationa. Flavin mononucleotide riboswitchb. Found in 5’ untranslated region of ribDEAHT operon: Riboflavin biosynthesis proteinsc. Riboswitches sense many metabolites including amino acids (glycine, lysine), vitamins (cobalamin, thiamin, SAM) and purines (adenine, guanine)d.13. Aptamer domain structures bind metabolites tightly and are mutually exclusive with terminator
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