Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter

Abstract

We describe the characterization in vitro of three hybrid trp-lacUV5 (TAC) promoters, which have perfect homology to the -10 and -35 Escherichia coli promoter consensus hexamer sequences, but which differ in the distance between the -10 and -35 regions. The three promoters, TAC16, TAC17, and TAC18 have spacings of 16, 17, and 18 base pairs, respectively. We have measured KB and k2, the constants that describe the formation of open complexes, on these three promoters. We have also measured their relative strengths on supercoiled plasmid DNA. Our results show that these are the strongest promoters that have been characterized in vitro so far and confirm the hypothesis that the consensus promoter sequence is "best." We find the TAC17 promoter (KBk2 = 8.4 X 10(7) M-1 s-1) to be stronger than either the TAC16 (KBk2 approximately 1.5 X 10(7) M-1 s-1) or TAC18 (KBk2 approximately 3.5 X 10(7) M-1 s-1) promoters, a result that is in agreement with other findings on the effect of spacer length. The choice of start point for transcription is affected by spacer length. Transcription from all the promoters was stimulated at moderate concentrations of salt (less than 150 mM) and persisted at high salt concentrations (300 mM).