Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter.
The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C. Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).
Cowing
D W
DW
Department of Bacteriology, University of Wisconsin-Madison 53706.
Mecsas
J
J
Record
M T
MT
Gross
C A
CA
D001426:Bacterial Proteins / Q000235:genetics; D018833:Chaperonins; D003596:Cytosine; D004269:DNA, Bacterial / Q000648:ultrastructure*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006147:Guanine; D006360:Heat-Shock Proteins / Q000235:genetics*; D066298:In Vitro Techniques; D008745:Methylation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism*; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; D006147:Guanine; D003596:Cytosine; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D018833:Chaperonins