In vitro characterization of hybrid promoters and altered tryptophan operon promoters.

Abstract

This study examines the in vitro interaction of hybrid and altered Escherichia coli promoters and other promoters with purified E. coli RNA polymerase. Three parameters of polymerase activity were examined: the time for open complex formation; the temperature of transitions; and the time required for productive initiation. The results indicate the rate of in vitro binding as measured by the filter binding technique does not completely correlate with the in vivo activities among these diverse promoters. Transition temperatures ranged from 13 to 27 degrees C with the lowest transition temperatures associated with the relatively weak in vivo beta-lactamase and anti-tet promoters. The productive initiation studies showed a dependence on labeled nucleoside triphosphate concentration when that nucleotide was present early and frequently in the transcript. Promoters containing the -10 region of the lac promoter had slow productive initiation rates while trp -10 promoter derivatives were generally very fast. In the promoters studied here, a trend was noted between the binding rate and transition temperature studies in that the promoters with the lower transition temperatures tended to bind more rapidly.