Sequence-Dependent Upstream DNA–RNA Polymerase Interactions in the Open Complex with λPR and λPRM Promoters and Implications for the Mechanism of Promoter Interference

Received 23 July 2008; received in revised form 11 November 2008; accepted 12 November 2008 Available online 24 November 2008 Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the PR and PRM promoters of bacteriophage λ have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at PR [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex.EMBO J., 18, 4464–4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from −36 to −59 and from −80 to −100. Likewise, RPos formed at PRM showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of αsubunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNAwrapping but has only a small effect on the activity of the PR promoter. We find, however, that the frequency of DNA templates with both PR and PRM occupied by an RNAP significantly increases upon loss of DNAwrapping. These results suggest that α carboxyterminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between PR and PRM is proposed.