Probing the assembly of transcription initiation complexes through changes in sigmaN protease sensitivity.
The alternative bacterial sigmaN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of sigmaN, the assembly of end-labeled Klebsiella pneumoniae sigmaN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free sigmaN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that sigmaN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.
Casaz
P
P
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London, SW7 2BB, United Kingdom.
Buck
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D007711:Klebsiella pneumoniae; D010449:Peptide Mapping; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics; D051877:RNA Polymerase Sigma 54; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D011494:Protein Kinases; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54