Stevens A M, Greenberg E P
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
J Bacteriol. 1997 Jan;179(2):557-62. doi: 10.1128/jb.179.2.557-562.1997.
LuxR is required for cell density-dependent activation of the Vibrio fischeri luminescence (lux) genes. It has not been possible to study full-length LuxR in vitro, but a polypeptide containing the C-terminal transcriptional-activator domain of LuxR (LuxRdeltaN) has been purified, and its binding to lux regulatory DNA has been investigated. By itself, LuxRdeltaN interacts with a region of lux regulatory DNA that is upstream of the lux box, which is a 20-bp element that is required for LuxR activation of the luminescence operon. Individually, neither the purified LuxRdeltaN nor RNA polymerase binds to the lux box region, but together the two proteins bind in synergy to the lux box-luxI promoter region. We show that binding of LuxRdeltaN to the upstream region is not a prerequisite for its synergistic binding with RNA polymerase to the lux box and the luxI promoter region. We also show that LuxRdeltaN and RNA polymerase are both required and sufficient for transcriptional activation of the lux operon. This argues against the hypothesis that LuxR functions to alleviate repression of the lux operon by another cellular factor. Rather, our data support the view that LuxR functions as an accessory factor that enables RNA polymerase to bind to and initiate transcription from the promoter of the lux operon.
LuxR是费氏弧菌发光(lux)基因的细胞密度依赖性激活所必需的。目前尚无法在体外研究全长LuxR,但已纯化出一种包含LuxR C端转录激活结构域的多肽(LuxRdeltaN),并对其与lux调控DNA的结合进行了研究。单独来看,LuxRdeltaN与lux调控DNA中位于lux框上游的一个区域相互作用,lux框是一个20个碱基对的元件,是LuxR激活发光操纵子所必需的。纯化后的LuxRdeltaN和RNA聚合酶单独都不与lux框区域结合,但这两种蛋白质一起能协同结合到lux框-luxI启动子区域。我们发现,LuxRdeltaN与上游区域的结合并非其与RNA聚合酶协同结合到lux框和luxI启动子区域的先决条件。我们还表明,LuxRdeltaN和RNA聚合酶对于lux操纵子的转录激活都是必需的且足够的。这与LuxR通过另一种细胞因子减轻对lux操纵子的抑制作用这一假说相悖。相反,我们的数据支持这样一种观点,即LuxR作为一种辅助因子,使RNA聚合酶能够结合到lux操纵子的启动子并从该启动子起始转录。