EFFECT OF REVERSED ORIENTATION AND LENGTH OF A(N)CENTER-DOT-T-N DNA BENDING SEQUENCES IN THE -35 AND SPACER DOMAINS OF A CONSENSUS-LIKE ESCHERICHIA-COLI PROMOTER ON ITS STRENGTH IN-VIVO AND GRASS STRUCTURE OF THE OPEN COMPLEX IN-VITRO

In continuation of an earlier study (Lozinski et al., 1991 Nucleic Aci ds Res. 19, 2947-2953) a series of consensus-like E. coli promoters wi th bending A(n) . T-n sequences of different length (n = 3-8) and orie ntation in the 35 and spacer domains was constructed, cloned into the plasmid pDS3 and their strength in vivo measured in relation to an int ernal transcriptional standard. Gel mobilities of free DNA restriction fragments carrying these promoters and of open transcriptional comple xes with cognate RNA polymerase were determined by polyacrylamide gel electrophoresis and the gross structure of the complexes interpreted i n terms of the theoretically predicted superstructure of DNA restricti on fragments. The results obtained together with those reported earlie r show that bending of the DNA helix axis immediately upstream of the 35 domain generally lowers the promoter strength in vivo and brings ab out shortening of the mean square end-to-end distance between free DNA ends in the open complex in vitro. T-4(-34...-37) and T-5(-34...38) t racts located in the nontemplate DNA strand had the largest and compar able effect on the promoter strength, while the a A(5) . T-5 (-37...-4 1) sequence in either orientation (As tract in the template or nontemp late strand) exerted a much smaller effect. Promoters with the spacer bent by about 40 degrees but in different directions, by two An (n = 5 or 4) tracts aligned in phase with the B-DNA repeat and located eithe r in the template or nontemplate strands; had somewhat lower strength irt vivo but the gross geometry of the respective open complexes was t he same as that of a control promoter with straight spacer. Implicatio ns of these findings are discussed in connection with the existing mod el of E. coli transcriptional open complex.