Prokaryotic Transcription Mechanism Flashcards
basics of transcription
promoter specifies TSS: consists of cis elements - shortly upstream of TSS recognised by RNA Pol
2 phases: initiation, elongation, termination. 5’ tp 3’. NTP substrates. no primer. NMP incorporated, PPi released
what does the NAD+ cap do
at 5’ end. stabilises prokaryotic mRNAs. NAD+ contains ADP-ribose-nicotinamide. only occurs at some promoters: seq of promoter important.
NAD+ can compete w ATP for direct incorporation by RNA pol at +1 position. can regulate.
Structure of RNA Polymerase (RNAP)
single core RNAP synthesises mRNA, rRNA, tRNA.
Holo-enzyme: accurate initiation + efficient elongation
Core enzyme: efficient elongation, but multiple start sites
alpha2 (assembly) ,[ beta ‘, beta] active site , - core enzyme.
holo enzyme includes this omega, sigma (confers promoter recognition)
cleft surrounded by flexible pincers/jaws. Jaws enclose downstream DNA in elongation complex. active site is located at base of large cleft formed between beta and beta prime subunits.
cleft flanked by mobile pincers/jaws enclose 20 bp downstream DNA in elongating transcription complex.
N-terminal domains of alpha in structure. CTD of alpha connected to NTD by flexible linker and can bind DNA.
how does RNAP find promoters correctly?
consensus sequence: -10 or Pribnow Box. elements w high match to consensus are functionally stronger, low match weaker.
1. Can align multiple sequences to find consensus.
- examine natural promoter mutations - affect quantity but not seq of mRNA. either UP or DOWN mutations higher lower levels of mRNA. strong promoter matching consensus seq - DOWN mutations more common. Weak = UP.
- Generate targeted mutations - mutations experimentally.
- Biochem mapping: DNA-protein interactions, DNA melting (KMnO4 oxidation of ss thymidines)
what are 4 methods to map DNA-protein interactions
- Electrophoretic mobility shift assay (EMSA)
- Footprinting
- Modification interference
- Chromatin immnoprecipitation (ChIP)
how does EMSA work
does protein interact w fragment of DNA? - need labelled probes w mutations or ability or WT and mutant unlabelled to compete for binding
31P end-labelled DNA, incuble w pure DNA binding protein or cell extract
Non-denaturing gel electro - autoradiography or imager.
sequence specificity demonstrated by adding excess unlabelled specific or non-spec (point mutation) DNA
how does footprinting work
where does protein binding protect DNA from attack by nucleases or chemical probes
i. 32P end-labelled DNA (only one strand labelled)
ii. incubate +/- protein
iii) Digest mildly
iv) High res Denaturing (6M urea) gel electro & Autoradiography
digest by DNase (seq non-specific), OH radicals (seq non-spec), DMS heat treatment depurinates MR bases, alkali cleaves backbone at depuri sites, is cell permeable so either vivo or vitro.
how does modification interference work
what positions does prior chem modification of DNA prevent protein-binding
i) 31P end-labelled DNA(only one strand labelled)
ii) Modify DNA – average 1 modification per strand (DMS)
iii) Incubate DNA with protein
iv. separate bound from unbound DNA (gel shift)
v. purify DNA and chemically cleave modified sites
vi. denaturing gel
missing bands where modification prevents protein binding
How does Chromatin immunoprecipitation (ChIP)
monitors DNA sites at which spec proteins bind in vivo. low res but can genome wide. cross links - proteni-protein and protein nucleic. can probe DNA binding in vivo
i) Treat live cells (i.e. in vivo) with formaldehyde: H2CO
ii) Lyse cells, sonicate to break DNA to ~200 bp fragments
iii) Use specific antibody to immunoprecipitate DNA fragments
bound to protein of interest
iv) Heat-treat to reverse crosslinks, then purify DNA
v) quantitative real time PCR for DNA regions of interest (e.g. promoters)
Or:
Deep sequencing (ChIP-Seq) to globally profile protein binding to genomic DNA
describe bacterial core promoter elements
-10 and -35 2 consensus sequences - match determines of transcriptional efficiency. sequences asymmetric - no repeats and recog by RNAP sigma subunit.
-10/Pribnow Box:TATAATG (melted non-template strand recognized by sigma region 2, DOWN mutation inhibit promoter melting)
-35 box: TTGACA recog by sigma region 4, DOWN mut inhibit initial binding
UP element at highly active rRNA promoters: 20bp A/T rich upstream -35. recog CTD of alpha subunits. binds via minor groove.
How RNAP binding to Promoter DNA
core enzyme: binds DNA non-spec (loose binding), non-spec transcription in vitro, initiation from multiple locations on both strands
Holo enzyme (+ sigma): non spec binding reduced by 10^3/4. promoter affinity increased (more efficient), accurate initiation of transcription
what are the domains of the sigma subunit
domain 2 + 4 contain helix-turn helix motifs that recognises -10 and -35 boxes but sigma alone does not bind DNA.
Domain 1.1 neg charged “DNA mimic” - suppresses inappropriate DNA binding
in free sigma, 1.1 interacts w s4 - prevents DNA binding so specific promoter recog only enabled when part of holoenzyme
in holoenzyme, s1.1 occupies downstream DNA binding cleft, reducing non-spec DNA binding - interaction is displaced upon promoter binding
sequence of events during initiation:
closed and open complexes, abortive initiation & promoter escape
open: First NTP binds w low affinity by base-pairing to template strand. subsequent greater affinity. phosphodiester bonds formed between adjacent NTPs. transcription bubble expands in 3’.
multiple cycles of abortive initiation collapse of bubble - if obstacles to elongation, interaction not stable.
