The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase.
BACKGROUND
The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure alpha2betabeta') associated with one of the sigma subunits, required for promoter recognition. Different sigma factors compete for core binding. Among the seven sigma factors present in E. coli, sigma70 controls gene transcription during the exponential phase, whereas sigmaS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled sigmaS and sigma70, we compared the affinities of both sigma factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes.


RESULTS
Using native polyacrylamide gel electrophoresis, we demonstrate that sigmaS binds to the core enzyme with fivefold reduced affinity compared to sigma70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of sigmaS, while increasing the accessibility in region 4.1 of sigmaS. We have also analysed the positioning of sigmaS on the holoenzyme by the proximity-dependent protein cleavage method using sigmaS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the beta and beta' subunits, but not alpha. Regions 2.5, 3.1 and 3.2 of sigmaS are close to both beta and beta' subunits, in agreement with iron chelate protein footprinting data.


CONCLUSIONS
A comparison between these results using sigmaS and previous data from sigma70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.
Colland
Frédéric
F
Institut Pasteur, Laboratoire des Regulations Transcriptionnelles (FRE 2364 CNRS), 75724 Paris Cedex 15, France.
Fujita
Nobuyuki
N
Ishihama
Akira
A
Kolb
Annie
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D023302:Protein Footprinting; D020539:Sequence Analysis, Protein; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012808:Sigma Factor; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine