The Prokaryotic Transcription Apparatus Flashcards
Central Dogma
DNA->RNA->Protein
What transcribes DNA into RNA
RNA polymerase
What does RNAP require?
DNA template, 4 ribonucleotide 5’ triphosphate, Mg2+
What is a sigma factor? What is its role?
sigma sub-units control promoter choice; helps RNA polymerase to find primer and and helps open up promoter sequence.
Shine-Delgarno
sequence that acts as the ribosome binding site (RBS)
Termination sequence in prokaryotic genes
poly U’s
Rudder
piece of the RNA polymerase that is responsible for unzipping dsRNA as RNA polymerase is moving. Remember DNA polymerase requires a helicase. RNAP does not.
What is the function of the ASP residues in RNAP and DNAP?
They hold onto mg2+
What is the role of Mg2+ in RNAP and DNAP?
causes 3’OH on RNA growing strand to become a nucleophile and attack alpha phosphate on incoming rnTP, leaving of ppi causes favorable reaction.
What is the coding strand?
The coding strand is the non template strand. It’s sequence is the same as the growing mRNA, except that there are T’s instead of U’s
How long are transcription bubbles?
17 bp
Non-template strand
a.k.a coding or sense strand. Same as the RNA transcript, except for the replacement of T with U.
Numerical values: Transcription initiation site, downstream, and upstream
Transcription initiation site = +1 ; Downstream = +2,3,4…. (direction of transcription) ;
Upstream : -1,2,3,……
How do we MAP promoters?
1) Radiolabel DNA with possible promoter sequence 2) divide radiolabeled DNA into two tubes 3) 1 tube DNAP another w/O DNAP 4) Add DNAase (Cuts only occur in regions that don’t have proteins bound to it i.e RNAP or DNAP) 5) missing bands indicate where DNAP or RNAP bound to DNA 6) Sequence uncut regions.
Consensus sequence
Region where sigma 70 binds to. -35 region 16-19 bp spacer, pribnow box
-35 region
TTGACA
Pribnow box
TATAAT -10 region
Spacer sequence
16-19bp. Between -35 and -10 regions. absolute sequence is not important, but the length is important.
sigma 70
Housekeeping sigma in E.coli
Sigma 32
Heat shock sigma. Helps transcribe heat shock proteins.
Core Enzyme
RNAP w/o sigma factor. No specific promoter binding ; tight non-specific DNA binding ; weak polymerase activity.
Holoenzyme
RNAP w/ sigma factor. Binds specific promoters ; weak non-specific DNA binding ; Finds promoter 10,000 times faster ; high polymerase activity.
What does the Sigma factor (70) do?
Associates with the -35 and -10 region through different sub-domains, wraps along RNAP and associates with DNA.
Promoter Clearance
displacement of the the sigma 3/4 loop through the RNA exit channel allows RNAP to break free from the promoter and initiate transcription.
