Department of Biochemistry, University of Wisconsin - Madison, Madison, WI 53706, United States.
Biophysics Program, University of Wisconsin - Madison, Madison, WI 53706, United States.
J Mol Biol. 2022 Jul 15;434(13):167621. doi: 10.1016/j.jmb.2022.167621. Epub 2022 May 6.
An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λP promoter complexes. Strong position-dependences of overall rate constants (k/K analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.
为了理解基因表达的调控并推动从抗生素发现到启动子设计等领域的发展，我们需要了解细菌转录起始的动力学和机制。最近，已经确定了大肠杆菌 RNA 聚合酶（RNAP）-λP 启动子复合物的起始和 RNA-DNA 杂交生长的逐步正向动力学和机制，该过程通过省略焦磷酸（PPi）副产物而不可逆。由于杂交生长与启动子接触的破坏、泡关闭和 RNAP 逃逸的耦合，每个核苷酸添加步骤的总速率常数（k/K 类似物）的位置依赖性很强。在这里，我们研究了在细胞指数生长期中发现的 PPi 浓度下这些步骤（焦磷酸水解）的逆转。我们量化了 [PPi] 对长（>10 个核苷酸，出泡后）和短（stalled，夭折）RNA 合成量和速率的影响，以确定 PPi 如何调节起始。生理 [PPi] 使尿嘧啶掺入和其他一些起始步骤具有显著的可逆性。生理 [PPi] 降低了具有生产性起始的 RNAP-启动子复合物的分数和每个生产性复合物的 RNA 合成速率，同时增加了夭折起始复合物的分数，影响夭折率，并将夭折-生产分布转移到更短的 RNA 上。由于起始中强烈偏向于预迁移状态，一些起始复合物的焦磷酸水解速率比从延伸复合物中去除相同核苷酸的速率大几个数量级，并且对核苷酸身份（嘧啶>嘌呤）的依赖性更强。由于指数期细胞中的细胞质 [PPi] 比静止期细胞高得多，因此 [PPi] 对起始速率和 RNA 合成量的影响必须具有生理相关性。
