DNA-binding domain mutants of sigma-N (sigma(N), sigma(54)) defective between closed and stable open promoter complex formation

被引:18
作者
Oguiza, JA [1 ]
Buck, M [1 ]
机构
[1] UNIV LONDON IMPERIAL COLL SCI TECHNOL & MED, DEPT BIOL, LONDON SW7 2BB, ENGLAND
基金
英国惠康基金;
关键词
D O I
10.1046/j.1365-2958.1997.5861954.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The sigma(N) 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 sigma(N) C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigma(N) function. Mutants defective in supporting sigma(N)-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 sigma(N) 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 sigma(N) are discussed.
引用
收藏
页码:655 / 664
页数:10
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