Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes.
The sigma(S) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of Esigma(S) RNA polymerase, using sigma(S) derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of Esigma(S) open complexes does not depend on the nature of the promoter and is similar to that obtained with Esigma(70), but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to sigma(70), region 2.1 of sigma(S) appears to be far from DNA. Region 4.2 of sigma(S) appears less accessible than its counterpart in sigma(70) and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the -35 promoter element. This provides a structural basis for the minor role of the -35 sequence in sigma(S)-dependent promoter recognition.
Colland
F
F
Institut Pasteur, Unité de Physico-chimie des Macromolécules Biologiques (URA 1773 du CNRS), 75724 Paris Cedex 15, France.
Fujita
N
N
Kotlarz
D
D
Bown
J A
JA
Meares
C F
CF
Ishihama
A
A
Kolb
A
A
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D009942:Organometallic Compounds; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine