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York University
BIOL 2020
K Wheaton

What are antibiotics? Rifampicin? Potent chemotherapeutic agents, antibiotic inhibitor of bacterial RNA synthesis effective for treating uberculosis Requirements for RNA polymerase (3) 1. Template, DNA template, coding-sense (+), template-antisense (-) 2. Activated precursors, ribonucleoside triphosphates (ATP, GTP etc) 3. Divalent metal ion (cofactor) Mg 2+ or Mn 2+ RNA polymerase reaction , synthesis Direction of synthesis 5’3’. Elongation direction? Elongation? Driven by? similar to 3’-OH group at the terminus of growing chain attacks inner most phosphoryl group of incoming ribonucleoside triphosphate. Driven forward by hydrolysis of pyrophosphate (doesn’t require a primer) Describe initiation 5 subunits, holoenzyme (alpha2beta2sigma) sigma finds transcription site, initiation of synthesis then dissociates. RNA without this is called the core enzyme. Promoters (DNA sequences) direct RNA polymerase to proper site, DNA sequence are present on teh 5’ upstream side (10 site/pribnow box) and (35 sequence) nucleotide # from start site. 35 and 10 sequence , have the strongest TTGACA, TATAAT. Promoters, weak prmoters what? Mutation can cause? also tend to have multiple substitutions at these site. Diminish promoter activity. Sigma functions? Decreases affinity of RNA polymerase by a factor of 10^4, allows DNA to slide along it, core enzyme binds tightly to DNA, also enables RNA polymerase to recognize promoter sites, subunit released when the new RNA chain (nascent chain) reaches nine, after release it can now start initiation by another core enzyme. Catalytically How does the sigma assist the polymerase? Finds the correct promoter site and then Bacteria? dissociates to help another. Usually sigma70 (mass 70kd), other factors protect it from environmental conditions Bound RNA polymerase does what? In Unwinds a segment of DNA, 17bp, doesn’t bacteria? need a primer, bacteria: new RNA strands have a tag on the 5’ end (pppG or pppA, confirming that it grows in the 5’3’ end) Describe elongation, transcription bubble. Region containing RNA polymerase, DNA and Error rate? nascent RNA. Newly made RNA strand forms temporary hybrid helix with DNA (8bp), transcription bubble moves 170 A in a second, makes 50 nucleotides per second. Error: one mistake per 10^4/10^5 nucleotides, higher than that of DNA replication Describe termination. Stop signal? Formation of phosphodiester linkages stops, RNA-DNA hybrid dissociates, melted region of DNA reanneals, RNA pol releases. Palindrome : GC rich region followed by a sequence of T residues. RNA of this would be self complementary (forms hairpin structure), followed by 4 or more uracil residues. RNA polymerase pauses, RNA-DNA hybrid is unstable (weakest of rU-dA weakest of all) this hybrid dissociates, DNA template reforms double helix. Bubble closes Antibiotic inhibitors of transcription, Inhibits initiation of RNA synthesis, interferes rifampicin does? with formation of first few phosphodiester linkages in teh RNA chain, blocks channel which the RNA-DNA hybrid generated must pass. This happens by binding to a pocket in the channel and blocking the elongation Regulation of lactose metabolism? Helps with? Lac operon, E coli can use lactose for energy, Creates? enzyme of it (beta galactosidase) changes it to its 2 monomers, 2 proteins synthesized: galactoside permease and thiogalactoside transacetylase. Helps with adaptation to different environments. Describe lac operon Genes encoding enzymes for lacose are controlled by a single mechanism, structural genes/regulatory genes, regulator gene encodes a repressor protein that binds to the operator site, includes all the enzymes from ^ Repressor I, (repressor), 0 (operator site), when it binds it prevents the 3 genes from being transcribed, (z, y, a) galactosidase, permease, transacetylase. Also has promoter site (p), correct transcription initiation site. Absence of lactose, repressor binds tightly and rapidly, blocking bound RNA pol from using DNA as a template. Inducer Binds to lac repressor, reducing its affinity for operator DNA, repressor-inducer complex leaves the DNA and allows transcription of operon. How is inducer induced? What else induces By allolactose (combo of galactose and beta galactosidase? glucose with alpha 1-6 rather than alpha 1-4 linkage. It is a side product of beta galactosidase reaction produced from the low levels of the molecule before induction. IPTG, potent inducer of the expression of the beta galactosidase, used in the lab as gene express. Prokaryote gene expression 3 promoter elements, primary transcript serves as mRNA, used immediately for protein synthesis, do not extensively modify RNA, translation and transcription are closely coupled, translation happens during transcrip. Eukaryote gene expression 1. RNA synthesis relies on promoter sequences in DNA to regulate initiation of transcription. Complexity comes from promoter elements 2. Modify RNA before it becomes mRNA,
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