Sun W, Hattman S
Department of Biology, University of Rochester, Rochester, NY, 14627-0211, USA.
J Mol Biol. 1998 Dec 11;284(4):885-92. doi: 10.1006/jmbi.1998.2228.
Transcription of the Mu mom operon requires activation by the phage gene product, C, a site-specific DNA binding protein. Previous in vivo and in vitro footprinting studies showed that Escherichia coli RNA polymerase (Esigma70=RNAP) bound the wild-type (wt) mom promoter (Pmom) region in the absence of C; this site, now designated momP2 (-11 to -64), is slightly upstream of, but overlapping with, momP1 (+16 to -49), the functional binding site for mom operon (rightward) transcription. The location/distribution of KMnO4-sensitive sites on the two DNA strands suggested that RNAP bound at momP2 was in an open-complex, but that transcription was in the opposite direction. Here, we used both runoff transcription and reverse transcriptase-primer extension sequencing to provide direct evidence that in the absence of C protein, RNAP carries out leftward transcription from momP2 both in vitro and in vivo. In addition, the 5' ends of these transcripts were mapped to the same upstream initiation site, -58G, relative to the initiation site of C-activated rightward transcription. We also present evidence that leftward transcription from momP2 requires RNAP recognition of an UP-element by the carboxyl-terminal domain of the alpha subunit.
Mu噬菌体mom操纵子的转录需要噬菌体基因产物C（一种位点特异性DNA结合蛋白）的激活。先前的体内和体外足迹研究表明，在没有C的情况下，大肠杆菌RNA聚合酶（Esigma70 = RNAP）结合野生型（wt）mom启动子（Pmom）区域；这个位点，现在命名为momP2（-11至-64），位于momP1（+16至-49，mom操纵子（向右）转录的功能性结合位点）的上游且与之重叠。两条DNA链上对高锰酸钾敏感位点的位置/分布表明，结合在momP2的RNAP处于开放复合物状态，但转录方向相反。在这里，我们使用了径流转录和逆转录酶-引物延伸测序来提供直接证据，证明在没有C蛋白的情况下，RNAP在体外和体内均从momP2进行向左转录。此外，相对于C激活的向右转录的起始位点，这些转录本的5'端被定位到相同的上游起始位点-58G。我们还提供证据表明，从momP2向左转录需要α亚基的羧基末端结构域对上游元件（UP-element）进行RNAP识别。
