title: Characterisation of holoenzyme lacking sigmaN regions I and II.
abstract: The sigma-N (sigmaN) protein associates with bacterial core RNA polymerase to form a holoenzyme that is silent for transcription in the absence of enhancer-binding activator proteins. Here we show that the acidic Region II of sigmaN from Klebsiella pneumoniae is dispensable for polymerase isomerisation and trans-cription under conditions where the inhibited state of the holoenzyme is relieved by removal of sigmaN Region I sequences. Holoenzymes lacking Region I or Regions I+II were equally susceptible to the order of addition-dependent inhibition or stabilisation of DNA binding afforded by in trans Region I sequences. Region I+II-deleted [sigma] formed a holoenzyme with a DNA-binding activity more susceptible to inhibition by non-specific DNA than that lacking Region I. Region II sequences appear more closely associated with formation of a holoenzyme and [sigma] proficient in DNA binding than with changes in holoenzyme conformation needed for unmasking a single-strand DNA-binding activity used for open complex for-mation. Region II may therefore function to optimise DNA interactions for an efficient sigma cycle.
authors.affiliation: Department of Biology, Biomedical Sciences Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK.
authors.forename: W
authors.initials: W
authors.lastname: Cannon
authors.forename: M
authors.initials: M
authors.lastname: Chaney
authors.forename: M
authors.initials: M
authors.lastname: Buck
mesh_terms: D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D051877:RNA Polymerase Sigma 54; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D009692:Nucleic Acid Heteroduplexes; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D015719:Single-Strand Specific DNA and RNA Endonucleases