Dai X, Rothman-Denes L B
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Genes Dev. 1998 Sep 1;12(17):2782-90. doi: 10.1101/gad.12.17.2782.
Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present. We report that vRNAP-promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand. Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity. Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription. Eco SSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin. Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both. We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.
噬菌体N4编码的病毒体包裹的RNA聚合酶（vRNAP），当模板为超螺旋且存在大肠杆菌单链DNA结合蛋白（Eco SSB）时，能够结合并转录含启动子的双链DNA。我们报告，vRNAP对这些模板的启动子识别和活性需要模板链上的特定序列和发夹结构。由Mg(II)和生理超螺旋密度诱导的发夹挤出对于为聚合酶识别提供正确的DNA结构至关重要，因为不形成发夹的突变启动子在体外活性降低。因此，超螺旋诱导的DNA结构转变调节N4 vRNAP转录。Eco SSB通过稳定模板链发夹在生理超螺旋密度下激活转录。启动子处的特定序列是保守的，以提供与vRNAP的适当接触，支持发夹挤出，或两者兼而有之。我们提出了一个vRNAP启动子在体内利用的模型，并讨论了DNA超螺旋和Eco SSB在启动子激活中的作用。
