Kinetic and thermodynamic basis of promoter strength: Multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence
Kinetic and thermodynamic basis of promoter strength: Multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence
复制标题
DOI：
10.1021/bi0158472
复制
发表时间：
2002-03-19
期刊：
影响因子：
2.9
通讯作者：
Patel, SS
中科院分区：
文献类型：
--
作者：
Bandwar, RP;Jia, YP;Patel, SS
T7 RNA聚合酶（RNAP）的转录起始受特异性启动子DNA序列调节，该序列通常分为两个主要结构域，即结合结构域（-17至-5）和起始结构域（-4至+6）。这些结构域中非共有碱基的出现导致了启动子强度的多样性，其基础通过研究结合结构域的启动子特异性区域（-13至-6）和/或起始结构域的解链区域（-4至-1）的变化来研究T7启动子。瞬态动力学和热力学研究表明，转录起始途径中的多个步骤受到启动子DNA序列的调节。在天然phi3.8启动子中发现的启动子特异性区域中-11、-12和-13处的三个碱基变化使T7 RNAP对启动子DNA的总体亲和力降低2-3倍，并降低了第一种RNA产物pppGpG合成的速率。启动子开放在T7 RNAP中是化学驱动的。并且在解链区中的单个碱基变化（TATA至TAAA）降低了平衡时产生的开放复合物的程度。在解链区的碱基变化也增加了（+1）GTP的Kd和pppGpG的解离速率。因此，不同T7启动子的转录起始受起始GTP浓度的差异调节。T7启动子DNA的特异性和解链区既独立又协同地影响转录起始的不同步骤。虽然启动子序列变异对起始途径中的每一步都有很小程度的影响，但累积效应决定了整个启动子的强度。
Transcription initiation by T7 RNA polymerase M RNAP) is regulated by the specific promoter DNA sequence that is classically divided into two major domains, the binding domain (- 17 to -5) and the initiation domain (-4 to +6). The occurrence of nonconsensus bases within these domains is responsible for the diversity of promoter strength, the basis of which was investigated by studying T7 promoters with changes in the promoter specificity region (- 13 to -6) of the binding domain and/or the melting region (-4 to - 1) of the initiation domain. The transient state kinetics and thermodynamic studies revealed that multiple steps in the pathway of transcription initiation are modulated by the promoter DNA sequence. Three base changes in the promoter specificity region at -11, -12, and -13, found in the natural phi3.8 promoter, reduced the overall affinity of the T7 RNAP for the promoter DNA by 2-3-fold and decreased the rate of pppGpG synthesis, the first RNA product. Promoter opening is thermodynamically driven in T7 RNAP. and a single base change in the melting region (TATA to TAAA) decreased the extent of open complex generated at equilibrium. This base change in the melting region also increased the K-d of (+1) GTP and the dissociation rate of pppGpG. Thus, transcription initiation at various T7 promoters is differentially regulated by initiating GTP concentration. The specificity and melting regions of T7 promoter DNA act both independently and synergistically to affect distinct steps of transcription initiation. Although each step in the initiation pathway is affected to a small degree by promoter sequence variations, the cumulative effect dictates the overall promoter strength.