Effect of reversed orientation and length of An.Tn DNA bending sequences in the -35 and spacer domains of a consensus-like Escherichia coli promoter on its strength in vivo and gross structure of the open complex in vitro.

Abstract

In continuation of an earlier study (Loziński et al., 1991 Nucleic Acids Res. 19, 2947-2953) a series of consensus-like E. coli promoters with bending An.Tn sequences of different length (n = 3-8) and orientation in the -35 and spacer domains was constructed, cloned into the plasmid pDS3 and their strength in vivo measured in relation to an internal transcriptional standard. Gel mobilities of free DNA restriction fragments carrying these promoters and of open transcriptional complexes with cognate RNA polymerase were determined by polyacrylamide gel electrophoresis and the gross structure of the complexes interpreted in terms of the theoretically predicted superstructure of DNA restriction fragments. The results obtained together with those reported earlier show that bending of the DNA helix axis immediately upstream of the -35 domain generally lowers the promoter strength in vivo and brings about shortening of the mean square end-to-end distance between free DNA ends in the open complex in vitro. T4(-34 ...-37) and T5(-34 ...-38) tracts located in the nontemplate DNA strand had the largest and comparable effect on the promoter strength, while the A5.T5(-37 ... -41) sequence in either orientation (A5 tract in the template or nontemplate strand) exerted a much smaller effect. Promoters with the spacer bent by about 40 degrees but in different directions, by two A(n) (n = 5 or 6) tracts aligned in phase with the B-DNA repeat and located either in the template or nontemplate strands, had somewhat lower strength in vivo but the gross geometry of the respective open complexes was the same as that of a control promoter with straight spacer. Implications of these findings are discussed in connection with the existing model of E. coli transcriptional open complex.