RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation
RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation
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DOI：
10.1021/acs.biochem.9b00049
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发表时间：
2019-05-07
期刊：
影响因子：
2.9
通讯作者：
Record, M. Thomas, Jr.
中科院分区：
文献类型：
--
作者：
Henderson, Kate L.;Evensen, Claire E.;Record, M. Thomas, Jr.
探讨大肠杆菌转录起始和逃逸的分步动力学和机制。coli RNA聚合酶从λ P-R启动子，我们量化的积累和衰减的瞬时短RNA中间体的启动子逃逸和全长（FL）RNA合成的途径在广泛的NTP浓度范围内的快速淬火混合和磷光成像分析凝胶分离。实验在19摄氏度下进行，其中几乎所有检测到的短RNA都是通过生产性复合物或非生产性（停滞）起始复合物中的终产物而不是来自流产起始的FL-RNA合成中的中间体。生产起始动力学数据的分析产生复合二阶速率常数的所有步骤的NTP结合和杂交延伸到逃逸点（11聚体）。这些速率常数中最大的是在不涉及DNA打开或易位的步骤中将UTP掺入二核苷酸pppApU。随后的步骤，其中每一个开始与可逆的易位和DNA开放，是较慢的速率常数变化超过10倍，解释为易位应力的影响易位平衡常数。合成4-和5-聚体、7-聚体至9-聚体和11-聚体的速率常数特别小，表明RNAP-启动子相互作用在这些步骤中被破坏。这些速率常数的减少与先前确定的类似于9千卡的成本从APR. Structural模型和先前的结果表明，三组小速率常数对应于连续中断的内裂，-10，和-35相互作用。讨论了T7 RNAP逃逸的相似性。
To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the lambda P-R promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 degrees C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined similar to 9 kcal cost of escape from APR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.