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Analysis of the architecture of the transcription factor σ N (σ 54 ) and its domains by circular dichroism
Author(s) -
Missailidis S.,
Can W. V.,
Drake A.,
Wang X. Y.,
Buck M.
Publication year - 1997
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1997.3691738.x
Subject(s) - circular dichroism , biology , transcription (linguistics) , dna , rna polymerase , microbiology and biotechnology , protein secondary structure , promoter , denaturation (fissile materials) , rna polymerase ii , dna binding domain , biophysics , transcription factor , crystallography , rna , biochemistry , gene expression , chemistry , gene , philosophy , linguistics , nuclear chemistry
Enhancer‐dependent transcription in bacteria requires the alternative transcription factor σ N (σ 54 ), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of σ N by circular dichroism (CD) analysis. The σ N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of σ N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36°C. The secondary structure displays a two‐state melting curve with a second Tm of 85°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 σ N , but not regions I or I + II, is required for the structural integrity of σ N at high pH. Measurements of pKa suggested that α‐helical structures are important in σ N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of σ N do not require regions I or I + II but were strongly diminished by C‐terminal truncation of σ N . Promoter DNA binding resulted in aconformational change in σ 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 σ N , the function of C‐terminal sequences, and domain communication and its role in activation of transcription are discussed.