Involvement of the sigmaN DNA-binding domain in open complex formation.
sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.
Oguiza
J A
JA
Department of Biology, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
Gallegos
M T
MT
Chaney
M K
MK
Cannon
W V
WV
Buck
M
M
D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D018983:DNA Footprinting; D004278:DNA, Superhelical / Q000235:genetics; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000235:genetics*; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D016962:Sinorhizobium meliloti / Q000235:genetics; D013463:Sulfuric Acid Esters; D013698:Templates, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D012808:Sigma Factor; D013463:Sulfuric Acid Esters; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D015719:Single-Strand Specific DNA and RNA Endonucleases; C007482:dimethyl sulfate