In vitro transcription of the Escherichia coli histidine operon primed by dinucleotides. Effect of the first histidine biosynthetic enzyme.

Abstract

Initiation of transcription of the Escherichia coli histidine (his) operon in vitro has been analyzed. The DNA of a specialized transducing phage, ø80dhis, was used as a template, and his RNA was measured by RNA/DNA hybridization. Taking advantage of the fact that E. coli RNA polymerase cannot initiate transcription when the nucleoside triphosphates are present at very low (5 muM) concentration, his RNA initiation was primed by dinucleoside monophosphates. It has been found that his RNA synthesis can be stimulated by one of the three dinucleotides CpA, ApA, and ApG. Under these conditions, it is the initiation of his RNA synthesis that is stimulated. Stimulation of his RNA synthesis by the three dinucleotides apparently occurs at a single initiation site, as judged by the nonadditivity of the effects of the three dinucleotides. This was further confirmed by the effect of phosphoribosyltransferase (the first enzyme of histidine biosynthesis, which specifically represses the synthesis of his RNA) on ApA primed RNA synthesis. Addition of his protein results in a sharp decrease of his RNA synthesis, with no effect whatsoever on the levels of RNAa transcribed from other regions of the template. Our data suggest that the 5' -terminal sequence of his RNA made in vitro is ApApG and that the base immediately preceding this sequence is C.