Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism

Molecular Microbiology
Sotiris MissailidisM Buck

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 structu...Continue Reading

Citations

Nov 5, 1999·Archives of Biochemistry and Biophysics·D J StudholmeM Buck
Feb 1, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Richard G SmithMichael R Price
Feb 8, 2000·The Journal of Biological Chemistry·D I SvergunM Buck
Aug 1, 2013·Molecular Microbiology·María A RendónMagdalene So
Oct 8, 2005·The Journal of Biological Chemistry·Michaeleen DoucleffDavid E Wemmer

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