Li T, Ho H H, Maslak M, Schick C, Martin C T
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-4510, USA.
Biochemistry. 1996 Mar 26;35(12):3722-7. doi: 10.1021/bi9524373.
T7 RNA polymerase recognizes a relatively small promoter extending only 17 base pairs upstream from the start site for transcription. A model for this recognition suggests that the enzyme interacts with the major groove of duplex DNA in the region centered at position -9 [Muller, D.K., et al. (1989) Biochemistry 28, 3306-3313], and recent kinetic analyses of promoters containing base analogs at positions -10 and -11 have provided support for this model [Schick, C., & Martin, C.T. (1993) Biochemistry 32, 4275-4280; Schick, C., & Martin, C.T. (1995) Biochemistry 34, 666-672]. In the current work, we extend this analysis across the proposed major groove, identifying specific base functional group contacts at positions -9 through -5. Specifically, the 6-carbonyl of guanine at positions -9 and -7, the 6-amino group of adenine at position -8, the 5-methyl group of thymine at position -6 and the 2-amino group of guanine at position -5 are identified as primary contacts. The results strongly support the model for duplex recognition in this region of the promoter and suggest that recognition continues along one face of the helix beyond the major groove and into the adjoining minor groove at position -5, where helix melting begins.
T7 RNA聚合酶识别一个相对较小的启动子，该启动子仅从转录起始位点向上游延伸17个碱基对。这种识别的一个模型表明，该酶与位于-9位置中心区域的双链DNA的大沟相互作用[Muller, D.K., 等人 (1989) 《生物化学》28, 3306 - 3313]，最近对在-10和-11位置含有碱基类似物的启动子的动力学分析为该模型提供了支持[Schick, C., & Martin, C.T. (1993) 《生物化学》32, 4275 - 4280; Schick, C., & Martin, C.T. (1995) 《生物化学》34, 666 - 672]。在当前的工作中，我们将这种分析扩展到所提出的大沟，确定了从-9到-5位置的特定碱基官能团接触。具体而言，-9和-7位置鸟嘌呤的6-羰基、-8位置腺嘌呤的6-氨基、-6位置胸腺嘧啶的5-甲基以及-5位置鸟嘌呤的2-氨基被确定为主要接触点。结果有力地支持了启动子该区域双链识别的模型，并表明识别沿着螺旋的一个面继续，超出大沟并进入-5位置相邻的小沟，在该位置螺旋开始解链。
