Regions of the Escherichia coli primary sigma factor sigma70 that are involved in interaction with RNA polymerase core enzyme.
BACKGROUND
The sigma factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most sigma factors share several regions with significant homology in their amino acid sequences (regions 1-4). Some primary sigma factors carry a large nonconserved segment between regions 1 and 2. The binding of an sigma factor to the core enzyme alters the structure and properties of the sigma factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli sigma70 by binding to core enzyme and promoter DNA.


RESULTS
A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals. Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced. In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared. The nonconserved acidic region of sigma70 in the holoenzyme became hypersensitive upon binding to DNA.


CONCLUSIONS
These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the sigma factor are involved in binding to the core enzyme. The nonconserved acidic region is likely to be more exposed by further binding of sigma factor to promoter DNA.
Nagai
H
H
Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka, Japan. hnagai@labstrg-1.lab.nig.ac.jp
Shimamoto
N
N
D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases