Activation of s28-dependent transcription by CRP 1109
medium. Activities are shown in Miller units (nmol ONPG
hydrolysed min-1 mg-1 dry cell mass), and are averages from
at least three independent experiments.
Primer extension
Transcript start sites were mapped by primer extension as
described in Lloyd et al. (2008), using RNA purified from
strain M182 carrying the aer200 promoter fragment cloned in
pRW50 and 5’ end-labelled primer D49724, which anneals
downstream of the HindIII site in pRW50. Primer extension
products were analysed on denaturing 6% polyacrylamide
gels, calibrated with sequencing reactions, and were visual-
ized using a Fuji phosphor screen and Bio-Rad Molecular
Imager FX.
Protein purification
Purified CRP protein was donated by David Grainger (Uni-
versity of Warwick, UK), and wild type E. coli core RNA poly-
merase was purchased from Epicentre Technologies
(Madison, WI). His-tagged RNA polymerase a subunits con-
taining a single cysteine residue at position 302 were pre-
pared and labelled with FeBABE as described by Lee et al.
(2003). FeBABE-tagged a subunits were incorporated into
core RNA polymerase using the reconstitution method of
Tang et al. (1995). Purified s28 and s70 proteins were prepared
from BL21(DE3) cells carrying the overexpression plasmid
pKXH100, as described by Grainger et al. (2008). Es28 and
Es70 holoenyzmes were made by mixing wild type or
FeBABE-labelled core RNA polymerase with an equimolar
amount of s28 or s70, and incubating for 20 min at room
temperature.
In vitro transcription assays
Caesium chloride preparations of pSR carrying the aer200
promoter fragment served as a template for multiple-round in
vitro transcription assays, as described by Savery et al.
(1998). 20 ng pSR/aer200 was incubated in transcription
buffer containing 40 mM Tris pH 7.9, 10 mM MgCl2, 1 mM
dithiothreitol, 100 mM KCl, 100 mg ml-1 bovine serum
albumin, 200 mM GTP, 200 mM ATP, 200 mM CTP, 10 mM UTP
and 5 mCi [a32P]-UTP. Where indicated, CRP was included at
100 nM and cAMP at 0.2 mM. Reactions were started by
adding Es28 or Es70. RNA products were analysed on a dena-
turing 5.5% polyacrylamide gel and visualized using a Fuji
phosphor screen and Bio-Rad Molecular Imager FX.
Footprinting and EMSA experiments
KMnO4 and FeBABE footprinting experiments were per-
formed on PstI-HindIII fragments prepared from caesium
chloride preparations of pSR carrying aer200. Fragments
were labelled at the HindIII end with [g-32P]-ATP using poly-
nucleotide kinase. KMnO4 footprints were performed follow-
ing the protocol of Browning et al. (2009) and FeBABE
footprints were carried out as described by Lee et al. (2003).
Each reaction contained approximately 3 nM labelled PstI-
HindIII DNA fragment in 20 mM HEPES pH 8.0, 5 mM MgCl2,
50 mM potassium glutamate, 1 mM DTT and 0.5 mg ml-1
BSA. KMnO4 footprinting reactions contained 0.2 mM cAMP,
100 nM CRP and 50 nM Es28 or Es70, as required. FeBABE
footprinting reactions contained 0.2 mM cAMP, 100 nM CRP
and 200 nM FeBABE-labelled Es28. The products of KMnO4
and FeBABE footprinting reactions were analysed on dena-
turing 6% polyacrylamide sequencing gels, calibrated with
Maxam-Gilbert ‘G + A’ sequencing reactions.
The EMSA experiments were performed using EcoRI-
HindIII fragments prepared from pSR derivatives, and end-
labelled using [g-32P] ATP and polynucleotide kinase. EMSA
reactions were carried out as described by Lloyd et al. (1998)
and were analysed on 5% polyacrylamide gels. Footprinting
and EMSA gels were visualized using a Fuji phosphor
screen, and analysed using a Bio-Rad Molecular Imager FX
and Quantity One software (Bio-Rad).
Acknowledgements
K.H. was supported by a PhD studentship from the UK
BBSRC and this work was funded by a Wellcome Trust Pro-
gramme Grant to S.J.W.B.
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© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 75, 1098-1111