title: Cooperativity and interaction energy threshold effects in recognition of the -10 promoter element by bacterial RNA polymerase.
abstract: RNA polymerase (RNAP) melts promoter DNA to form transcription-competent open promoter complex (RPo). Interaction of the RNAP σ subunit with non-template strand bases of a conserved -10 element (consensus sequence T-12A-11T-10A-9A-8T-7) is an important source of energy-driving localized promoter melting. Here, we used an RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with -10 element nucleotides. The results reveal a strong cooperation between RNAP interactions with individual -10 element non-template strand nucleotides and indicate that recognition of the -10 element bases occurs only when free energy of the overall RNAP -10 element binding reaches a certain threshold level. The threshold-like mode of the -10 element recognition may be related to the energetic cost of attaining a conformation of the -10 element that is recognizable by RNAP. The RNAP interaction with T/A-12 base pair was found to be strongly stimulated by RNAP interactions with other -10 element bases and with promoter spacer between the -10 and -35 promoter elements. The data also indicate that unmelted -10 promoter element can impair RNAP interactions with promoter DNA upstream of the -11 position. We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.
authors.affiliation: Department of Molecular Biology and Biochemistry, Waksman Institute of Microbiology Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA and Institutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mekler
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020029:Base Pairing; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D021901:DNA, Intergenic / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D020035:Holoenzymes; D046912:Multiprotein Complexes; D012321:DNA-Directed RNA Polymerases