Nucleic Acids Research, 1988, Vol. 16, No. 20 9789-9809
© 1988
Articles |
Visualization and quantitative analysis of complex formation between E.coli RNA polymerase and an rRNA promoter in vitro
Department of Bacteriology, University of Wisconsin 1550 Linden Drive, Madison, WI 53706, USA
Received March 17, 1988. Accepted September 15, 1988.
We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the Initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotlde production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polynerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 Is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.
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