Newlands J T, Josaitis C A, Ross W, Gourse R L
Department of Bacteriology, University of Wisconsin, Madison 53706.
Nucleic Acids Res. 1992 Feb 25;20(4):719-26. doi: 10.1093/nar/20.4.719.
Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.
大肠杆菌rrnB P1启动子的转录可通过一个顺式作用序列增强，该序列相对于转录起始位点在-35六聚体上游延伸至约-150处，即上游激活区（UAR）。UAR的激活涉及两个成分：（1）一种反式作用蛋白Fis，它与UAR中-60至-150之间的三个位点结合；（2）UAR序列本身，其独立于其他蛋白质影响RNA聚合酶（RNAP）的活性。我们将后者称为因子非依赖性激活（FIA）。除了与大肠杆菌启动子典型的-10和-35六聚体相互作用外，RNAP还与rrnB P1的-53区域接触，这可能与FIA效应有关。我们构建了一系列插入突变体，在Fis结合位点和-35区域之间的-46位含有整数和非整数螺旋圈数，并在体外和体内测量了所得启动子的活性。数据表明，Fis依赖性激活和因子非依赖性激活都依赖于螺旋面：Fis结合位点和负责因子非依赖性激活的序列必须相对于RNA聚合酶正确定向才能激活转录。这些结果与其他证据一起，支持了直接的Fis-RNAP相互作用参与上游激活的模型。我们还证明，即使这些序列位于RNAP结合位点上游，RNAP也与rrnB P1 UAR的-53区域相互作用，并且这些相互作用与因子非依赖性激活相关。
