Sun W, Hattman S
Department of Biology, University of Rochester, NY 14627, USA.
Nucleic Acids Res. 1996 Oct 15;24(20):4042-9. doi: 10.1093/nar/24.20.4042.
Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.
噬菌体Mu的mom操纵子转录需要噬菌体编码的C蛋白进行反式激活。DNA酶I足迹分析表明，在没有C蛋白的情况下，大肠杆菌RNA聚合酶E（σ）70（RNAP）结合到mom启动子（Pmom）区域的一个位点，即位于顶部（非转录）链上的P2位点（相对于转录起始位点从-64到-11）。该位点略位于P1位点（-49到+16）的上游，但与之重叠，P1位点是向右转录的功能性结合位点。C蛋白结合位点上游紧邻的三个GATC位点的宿主DNA [N6-腺嘌呤]甲基化，是防止大肠杆菌OxyR蛋白结合所必需的，OxyR蛋白在dam-菌株中会抑制mom转录。已知OxyR会诱导DNA弯曲，其在体内通常处于还原构象，但在标准体外条件下会转变为氧化状态。通过DNA酶I足迹分析，我们提供了证据支持以下提议：OxyR的氧化形式和还原形式在体外与它们的靶DNA序列相互作用方式不同。一种突变形式OxyR-C199S，在dam-宿主中能够在体内抑制mom表达。体外DNA酶I足迹分析表明，OxyR-C199S保护Pmom的顶部链从-104到-46区域，并产生还原型野生型OxyR的特征性保护模式。OxyR-C199S的预结合完全阻断了RNAP在无C蛋白时与P2位点的结合，而它仅轻微降低了C蛋白与其靶位点（DNA酶I足迹分析确定为-55到-28）的结合。相反，OxyR-C199S强烈抑制C激活的RNAP募集到P1位点。这些结果表明，OxyR的抑制作用是在C蛋白结合之后介导的。已经分离出一些突变，这些突变解除了对C激活的依赖性，并且与C激活的野生型启动子具有相同的转录起始位点。其中一个这样的突变体tin7，在-14处有一个单碱基变化，将一个T6序列变为T3GT2。OxyR-C199S在体外部分抑制了RNAP与tin7启动子的结合，尽管OxyR和RNAP-P1的结合位点可能不重叠，并且在dam-细胞中tin7的体内表达降低了5到10倍。这些结果表明，OxyR可以抑制tin7。
