BCH311H1 Lecture Notes - Lecture 8: Catabolite Repression, Transcription Factor Ii F, Chromosome
26 Aug 2018
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BCH311 – Lecture 8: Transcription I
Prokaryotes
• Transcription I
• Can reverse transcription with reverse transcriptase (RNA —> DNA) ; important
for retrovirus
• Proteins: complex of secondary and tertiary structures that help carry function
to facilitate cell survival
o Ex. transportation (Hb), enzymes, hormones
• “Central Dogma Of Molecular Biology”
o Linear progression of DNA —> RNA —> protein
• Overall Structure of the enzymes is quite similar
o Thus they have a common evolutionary ancestor
• RNA synthesis is in 3 stages: initiation, elongation, termination
• RNA Polymerase Functions:
o Initiation sites/Promoters-cis acting elements
▪ Synthesize strands de novo ( no primer required)
o Unwinds dsDNA
▪ Acts as helicase
o Correct ribonucleoside triphosphate selection unidirectional/processive
▪ Unidirectional and highly processive
o Termination
o Activation/Repression-transcription factors
▪ Promoter regions can bind these factors
o Fundamental Reaction
▪ Synthesizes phosphodiester bond
• RNA synthesis: 5’ to 3’
o No primer required
o No proofreading capability: error rate DNA (1 in 1010) RNA (1 in 105)
o Slower than DNAP (800 nt/s), RNAP (50 nt/s)
• E. coli RNAP
o 400 kDa, 4 different subunits
o Holoenzyme is α2ββ’σ70 (pentamer)
o α2ββ’ (core enzyme-contains the catalytic site)
▪ Catalytic site found in beta subunit
o σ decreases DNA binding affinity
o σ released after 10 nts are synthesized
Document Summary
Released after 10 nts are synthesized: rnap active site, similar to dnap, transition state of phosphodiester bond. Identification of promoter sequences used by rnap through footprinting: dsdna is labelled with 32p, 1. protein of interest that binds to rna (eg. rna pol, 2. No protein: use many test tubes and add dnase, dnase will create random sized fragments by creating nicks in dna, run sample on gel. Step 3: termination: termination, 1) ceasing of phosphodiester bond formation, 2) rna-dna hybrid dissociation, 3) rewinding of the dissociated dna strands, 4) release of dna by rna polymerase, what determines termination of transcription, 1. Protein dependent: termination signal, palindromic gc region, self complementary followed by a sequence of 4 or more u, protein independent. Intrinsic termination: termination sites consist of three structural features: Inverted repeats, rich in g:c, which form a stable stem-loop structure in. It moves along rna transcript, finds the (cid:862)tra(cid:374)s(cid:272)riptio(cid:374) (cid:271)u(cid:271)(cid:271)le,(cid:863) unwinds the dna:rna hybrid, and releases rna chain.
