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Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor σ32 at the groE promoter

1989; Elsevier BV; Volume: 210; Issue: 3 Linguagem: Inglês

10.1016/0022-2836(89)90128-9

1089-8638

Deborah W. Cowing, Joan Mecsas, M. Thomas Record, Carol A. Gross,

Genomics and Chromatin Dynamics

The interaction of Eσ32 with the groE promoter at temperatures between 0 °C and 37 °C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 °C Eσ32 fully protected sequences between −60 and −5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 °C the majority of the Eσ32 promoter complexes had a DNase I footprint almost identical with that seen at 37 °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 Eσ32 differed from those seen at 37 °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 °C and 27 °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 °C. Therefore we propose that the formation of an open complex by Eσ32 at the groE promoter involves three classes of steps: Eσ32 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. Eσ32 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).