title: Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex.
abstract: Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.
authors.affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA. Electronic address: mekler@waksman.rutgers.edu.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mekler
authors.affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.affiliation: Rowan University School of Osteopathic Medicine, Stratford, NJ 08084, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Borukhov
authors.affiliation: Public Health Research Institute Center, New Jersey Medical School, Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, NJ 07103, USA.
authors.forename: Arkady
authors.initials: A
authors.lastname: Mustaev
authors.affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA; Department of Biochemistry and Molecular Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA; Institutes of Gene Biology and Molecular Genetics, Russian Academy of Sciences, Leninsky Avenue, 14, 119991 Moscow, Russia. Electronic address: severik@waksman.rutgers.edu.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D055162:Biocatalysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000737:chemistry / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D061785:Transcription Initiation, Genetic
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C090751:dksA protein, E coli; D006159:Guanosine Tetraphosphate; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases
keywords: DNA–protein interactions; fluorescence spectroscopy; stringent response; transcription initiation; transcription regulation