title: A steady state assay for the RNA polymerase initiation reaction.
abstract: A new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction.
mesh_terms: D003090:Coliphages / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D013698:Templates, Genetic
chemical_list: D010618:Phenanthrolines; D011189:Potassium Chloride; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium