Development of RNA polymerase-promoter contacts during open complex formation.
We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2. A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.
Mecsas
J
J
Department of Bacteriology, University of Wisconsin-Madison 53706.
Cowing
D W
DW
Gross
C A
CA
D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005609:Free Radicals; D006878:Hydroxides; D017665:Hydroxyl Radical; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D004269:DNA, Bacterial; D005609:Free Radicals; D006878:Hydroxides; D017665:Hydroxyl Radical; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I