Correlating protein footprinting with mutational analysis in the bacterial transcription factor sigma54 (sigmaN).
Protein footprints of the enhancer-dependent sigma54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in sigma54 of potential functional importance at the interface between sigma54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of these positions. Properties of the mutant sigma54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of sigma54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other within core interacting sequences of sigma54, failed to stably bind the activator in the presence of ADP-aluminium fluoride, an analogue of ATP in the transition state for hydrolysis. Overall, the data presented describe a collection sigma54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the view that the interface between sigma54 and core RNAP is functionally specialised.
Wigneshweraraj
Siva R
SR
Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
Casaz
Paul
P
Buck
Martin
M
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D023302:Protein Footprinting; D017434:Protein Structure, Tertiary; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020035:Holoenzymes; C098954:PspF protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D001616:beta-Galactosidase