Lecture 5 1
Role of α subunit - α CTD
RNAP can recognize the upstream promoter element UP.
σ recognizes core promoter element (-10, -35);
What recognizes UP?
Gourse et al. –
mutations in a
subunit led to
DNA footprinting experiments
Wildtype RNAP footprints both core and UP element
A mutant RNAP footprints core promoter only Lecture 5 2
Therefore the α (C-terminal domain) is responsible for
interaction with the UP element.
Footprinting is a method for detecting protein-DNA
interactions that can tell where the target site lies on the
DNA in protein binding.
A protein bound on a DNA strand protects it from attack by
something that will degrade the DNA.
Therefore, it leaves
a “footprint” on the
Steps - DNAse:
end label DNA
(1 strand only)
Allow protein to
DNAse I under mild
1 cut per strand
strands, electrophorese beside untreated DNA.
DMS – chemical is smaller and reveals more subtleties; Lecture 5 3
Hydroxyl radicals are even smaller.
Analysis shows αNTD and αCTD fold independently to
form 2 domains tethered by a flexible linker.
Clevage reveals that alpha domains are linked by 13 aa
Promoters w/o UP are bound by σ only
UP promoters are bound by both σ and α
Stronger RNAP binding = stronger promoter
αCTD also interacts with activator proteins that bind to
sites outside of the core/extended promoter.
Also, αNTD plays a key role in helping organize RNAP
The order of subunit assembly is:
Alpha > alpha2 > alpha 2b > alpha beta beta prime
Hayward et al. -
Mutations of αCTD up to a certain point produced RNAP
that were still able to assemble.
With 94 AA’s removed, RNAP could assemble (more
NTD). BUT with 153 AA’s removed, no RNAP assembly
(less NTD). RNAP assembly lies in alpha NTD
Point mutations in the NTD that interact with β and β’ lie on
one side of the αNTD. Lecture 5 4
And this is the side that faces away from alpha CTD of the
alpha subunit dimer. Lecture 5 5
β involved in phosphodiester bond formation
Zillig 1970 – discovered a way to purify each individual