Sequences in σN determining holoenzyme formation and properties

被引:48
作者
Gallegos, MT [1 ]
Buck, M [1 ]
机构
[1] Univ London Imperial Coll Sci Technol & Med, Dept Biol, London SW7 2AZ, England
基金
英国生物技术与生命科学研究理事会;
关键词
sigma; 54; region I; RNA polymerase binding; heparin; 70;
D O I
10.1006/jmbi.1999.2704
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
Sigma subunits of bacterial RNA polymerases are closely involved in many steps of promoter-specific transcription initiation. Holoenzyme formed with the specialised minor sigma-N (sigma(N)) protein binds rare promoters in a transcriptionally inactive state and functions in enhancer-dependent transcription. Using competition and dissociation assays, we show that sigma(N)-holoenzyme has a stability comparable to the major sigma(70)-holoenzyme. Purified partial sequences of sigma(N) were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigma(N) which both bind the core but with significantly different affinities were identified, demonstrating that the sigma(N) interface with core RNA polymerase is extensive. The low affinity segment of sigma(N) included region I sequences, an amino terminal domain which mediates activator responsiveness and formation of open promoter complexes. The higher affinity site Lies within a 95 residue fragment of region III. We propose that the fore to region I contact mediates properties of the sigma(N)-holoenzyme important for enhancer responsiveness. Heparin is shown to dissociate sigma(N) and core, indicating that disruption of the holoenzyme is involved in the heparin sensitivity of the sigma(N) closed complex. (C) 1999 Academic Press.
引用
收藏
页码:539 / 553
页数:15
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