title: Sequence-dependent upstream DNA-RNA polymerase interactions in the open complex with lambdaPR and lambdaPRM promoters and implications for the mechanism of promoter interference.
abstract: Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P(R) and P(RM) promoters of bacteriophage lambda have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P(R) [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 P(RM) 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 alpha-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P(R) promoter. We find, however, that the frequency of DNA templates with both P(R) and P(RM) occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that alpha carboxy-terminal 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 P(R) and P(RM) is proposed.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Parma, Viale G. P. Usberti 23/A, 43100 Parma, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Mangiarotti
authors.forename: Sara
authors.initials: S
authors.lastname: Cellai
authors.forename: Wilma
authors.initials: W
authors.lastname: Ross
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
authors.forename: Claudio
authors.initials: C
authors.lastname: Rivetti
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid
chemical_list: D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase