BBMB 405 1st Edition Lecture 29Outline of Last Lecture XV. Chapter 29: RNA synthesis and ProcessingB. RNA polymerases catalyze transcription (con’t)Outline of Current Lecture XV. Chapter 29: RNA synthesis and ProcessingB. RNA polymerases catalyze transcription (con’t)C. Transcription in eukaryotes is highly regulatedCurrent LectureXV. Chapter 29: RNA synthesis and ProcessingB. RNA polymerases catalyze transcription (con’t)1. Metabolite sensing RNA structures called “riboswitches” can control terminationa. Riboswitches: as protein produced concentration increase, when reach a certain concentration then combine to riboswitch and turn off gene (feedback inhibition)b. Riboswitch: found in 5’ UTRs specific for that metabolite2. Rho (p) is an essential factor required for termination of ~half of E. coli genesa. In vitro transcription assay using E. coli RNAP holoenzymeb. Hexomeric, dependent on binding to ssRNAc. Not well sequenced, harder to predictThese 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.d. Rho binds then translocates until chatches up to polymerase, and dislodges RNA from DNAe. ATP dependent, used in 50% of E. coli genesf. Experiment: faithful transcription, except one long product, figured out that there is something not in original that controls length ; when add rho at different time then have different lengthsg. Rho Utilization Site (rut)h.3. Rho is a helicase that promotes RNA release from RNAPa. Binds at p sites (rut sites) – unstructured, C rich sequences downstream of stop codonb. Translocates to catch up to RNAP at transcription stop point (tsp) and stimulates RNA dissociationc.4. Antibiotics inhibit bacterial transcriptiona. Rifampicin- Binds ~ 12 Angstrums from active site- Blocks lengthening of RNA- Used to treat tuberculosis, leprosy, MRSA and others- Targets RNA polymerase, binds where hybrid is elongating and hybrid runs into antibiotic- If polymerase is already elongating protein then rifampicin can’t inhibititb. Phenoxazone- Actinomycin D- Inhibit transcription- Used to treat cancer- Binds to DNA through intercalation- In low concentrations it inhibits RNA polymerase- In large concentrations it is used in labs to test inhibition of replication (DNA polymerase)C. Transcription in eukaryotes is highly regulated1. Eukaryotic RNA transcription is complicateda. In a prokaryote nothing is compartmentalizedb. Eukaryotes: more complex, every RNA is processed, ribosome made in nucleolus and exportedc.2. Three different RNA polymerasesa. Not comprehensiveb. Focus on type IIc. Toxin alpha amanitin effects different polymerases3. Eukaryotic transcription initiation requires transcription factorsa. Analogous to sigma factors in prokaryotes (but much more complex): transcription factors may make up ~10% of protein-coding genes in human genomeb. Specific transcription factors recognize specific promoter sequences and recruit specific polymerasesc. Transcription factors have profound effects on cell differentiation, which is dependent on gene expression patterns: differentiated cells can be reprogrammed to another cell type by changing expression of transcription factors4. Eukaryotic RNA polymerases and their promotersa. Focus on polymerase IIb. Multiple elements within one promoter control expressionc. Core has four different regions that can vary so prediction is trickyd. TATA box is like -10 region5. Core promoters of RNA polymerase IIa. Core promoter elements are often found in Pol II promoters although not always togetherb. ~25% of human promoters contain TATA boxes,no consensus sequence makes it hard to identify promoter, if you sequence all of RNA you see that more is transcribed than predictedc. TFIIB recognition element (BRE) is often located just upstream of TATA boxes; complex of core transcription factors for polymerase IId. Downstream promoter elements (DPE) are located downstream of transcription start site and are often found in promoters lacking TATA boxes (although not always)6. Polymerase II promoters contain various regulatory elementsa. Cis (same strand/region)-acting elements: regulatory sequences affecting the same moleculeb. Enhancers, CAAT and GC boxes bind regulatory transcription factorsc. Mediator complex bridges transcription factors bound at regulatory elements and core promoter7. Transcription factors control transcription a. Promoters and cis regulatory elements are bound by transcription factorsb. General transcription factors are constitutively active (always on) and bind core promoter region8. TATA binding protein (TBP) is general transcription factora. Minor groove binding, mainly through hydrophobic interactionsb. Phe residues intercalate at ends of AT-rich sequencesc. DNA duplex is distorted, facilitating dsDNA unwindingd. TBP is part of TFIID complexe. Recognizes TATA boxes with high affinityf. Needs to recognize sequence, but doesn’t really do it; recognizes minor grove,but doesn’t make specific
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