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Abstract
Bacteriophage lambda promoters PR and PRM direct RNA synthesis in divergent orientations from start sites 82 base pairs apart. We had previously determined that the presence on the same DNA fragment of a wild type PR promoter interfered with the utilization of the PRM promoter. The results reported here concern the effects of changing the distance between the start sites by insertion or deletion of 5 or 10 base pairs. Three different techniques (run-off transcription, gel mobility shift, and permanganate probing) were employed to monitor complex formation at PRM. Unexpectedly we find that deletion of 10 base pairs between the start sites abolishes the interference, whereas insertion of 10 base pairs does not. Deletion of 5 base pairs, however, essentially prevents joint complex formation at PR and PRM. These findings suggest several ways in which for the wild type separation of the two promoters the utilization of PRM could be affected by an RNA polymerase at PR. In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase.
Highlights
Open complex formation at the wild type PR promoter occurs within seconds [6, 7], but formation of the complex at PRM is several orders of magnitude slower, on the time scale of tens of minutes [1, 3, 8]
To address the effect of RNA polymerase binding at PR on open complex formation at PRM for the fragment with wild type, I10, and D10 spacings, we made variants for which PR had been inactivated by a point mutation in the Ϫ10 region
We have shown by three independent assays that open complex formation at PRM benefits from a 10-base pair reduction in the distance between the PR and PRM promoters
Summary
Open complex formation at the wild type PR promoter occurs within seconds [6, 7], but formation of the complex at PRM is several orders of magnitude slower, on the time scale of tens of minutes [1, 3, 8]. To address the effect of RNA polymerase binding at PR on open complex formation at PRM for the fragment with wild type, I10, and D10 spacings, we made variants for which PR had been inactivated by a point mutation in the Ϫ10 region.
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