Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase.
A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered sigma70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA.Fe (FeBABE). RNA polymerase core reconstituted with tagged sigma derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of sigma with promoter DNA is discussed.
Bown
J A
JA
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Owens
J T
JT
Meares
C F
CF
Fujita
N
N
Ishihama
A
A
Busby
S J
SJ
Minchin
S D
SD
D001483:Base Sequence; D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics*; D006868:Hydrolysis; D007502:Iron Chelating Agents; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D004274:DNA, Recombinant; D007502:Iron Chelating Agents; D009942:Organometallic Compounds; D012808:Sigma Factor; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases