McLeod S M, Xu J, Johnson R C
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USA.
J Bacteriol. 2000 Aug;182(15):4180-7. doi: 10.1128/JB.182.15.4180-4187.2000.
The Escherichia coli proP P2 promoter, which directs the expression of an integral membrane transporter of proline, glycine betaine, and other osmoprotecting compounds, is induced upon entry into stationary phase to protect cells from osmotic shock. Transcription from the P2 promoter is completely dependent on RpoS (sigma(38)) and Fis. Fis activates transcription by binding to a site centered at -41, which overlaps the promoter, where it makes a specific contact with the C-terminal domain of the alpha subunit of RNA polymerase (alpha-CTD). We show here that Fis and cyclic AMP (cAMP) receptor protein (CRP)-cAMP collaborate to activate transcription synergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding to a site centered at -121.5, but CRP without Fis provides little activation. The contribution by CRP requires the correct helical phasing of the CRP site and a functional activation region 1 on CRP. We provide evidence that coactivation is achieved by Fis and CRP independently contacting each of the two alpha-CTDs. Efficient transcription in vitro requires that both activators must be preincubated with the DNA prior to addition of RNA polymerase.
大肠杆菌proP P2启动子可指导脯氨酸、甘氨酸甜菜碱及其他渗透保护化合物的整合膜转运蛋白的表达，在进入稳定期时被诱导，以保护细胞免受渗透压冲击。P2启动子的转录完全依赖于RpoS（σ38）和Fis。Fis通过结合位于-41位点中心且与启动子重叠的位点来激活转录，在该位点它与RNA聚合酶α亚基的C末端结构域（α-CTD）进行特异性接触。我们在此表明，Fis和环磷酸腺苷（cAMP）受体蛋白（CRP）-cAMP在体外协同激活转录。体内和体外的共激活都依赖于CRP结合到位于-121.5位点中心的位点，但没有Fis时CRP几乎没有激活作用。CRP的作用需要CRP位点正确的螺旋相位以及CRP上的功能性激活区域1。我们提供的证据表明，共激活是通过Fis和CRP分别独立接触两个α-CTD来实现的。体外高效转录要求在添加RNA聚合酶之前，两种激活剂都必须先与DNA预孵育。
