DNA-binding domain mutants of sigma-N (sigmaN, sigma54) defective between closed and stable open promoter complex formation

Molecular Microbiology
J A Oguiza, M Buck

Abstract

The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function. Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes. The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.

Citations

Feb 27, 2001·Nucleic Acids Research·S R WigneshwerarajM Buck
Jun 29, 1999·Cold Spring Harbor Symposia on Quantitative Biology·A KumarE P Geiduschek
Jul 14, 2000·Journal of Bacteriology·M BuckJ D Gralla
May 26, 2001·Biochemical and Biophysical Research Communications·D Kuczyńska-WisńikA Taylor
Mar 13, 2001·Journal of Molecular Biology·S R WigneshwerarajM Buck
May 18, 1999·Journal of Molecular Biology·M T Gallegos, M Buck
Jan 8, 1999·Journal of Molecular Biology·P Casaz, M Buck
Aug 14, 1999·Molecular Microbiology·J A OguizaM Buck

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