title: Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism.
abstract: Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of sigma N by circular dichroism (CD) analysis. The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C. The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C. The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting. In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting. Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH. Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N. Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant. A typical B-DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed.
authors.affiliation: Chemistry Department, University of York, Heslington, UK.
authors.forename: S
authors.initials: S
authors.lastname: Missailidis
authors.forename: W V
authors.initials: WV
authors.lastname: Cannon
authors.forename: A
authors.initials: A
authors.lastname: Drake
authors.forename: X Y
authors.initials: XY
authors.lastname: Wang
authors.forename: M
authors.initials: M
authors.lastname: Buck
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014443:Tyrosine; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54