A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases
A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases
复制标题
DOI：
10.1021/acs.biochem.8b01256
复制
发表时间：
2019-04-23
期刊：
影响因子：
2.9
通讯作者：
Schneider, David A.
中科院分区：
文献类型：
--
作者：
Scull, Catherine E.;Ingram, Zachariah M.;Schneider, David A.
真核细胞至少表达三种核RNA聚合酶(POL)，每一种都有一组独特的基因靶点。虽然这些酶是同源的，但POL之间有许多不同之处。在这项研究中，建立了一种新的Pol I转录延伸的检测方法，以探索Pol I之间的酶差异。在酿酒酵母中，触发环普遍保守的铰链区E1103G的突变会导致Pol II伸长率的功能增强，而Pol I的相应突变E1224G会导致功能丧失。E1103G Pol II突变稳定了触发环的闭合构象，促进了Pol II的催化步骤，这可能是Pol II的限速步骤。在单核苷酸和多核苷酸加成试验中，我们观察到E1224G Pol I降低了核苷酸加成速度和二核苷酸切割活性，增加了误掺入率。总而言之，这些数据表明，POL I至少在一定程度上受到与POL II相同步骤的速度限制，POL II是催化步骤。
Eukaryotic cells express at least three nuclear RNA polymerases (Pols), each with a unique set of gene targets. Though these enzymes are homologous, there are many differences among the Pols. In this study, a novel assay for Pol I transcription elongation was developed to probe enzymatic differences among the Pols. In Saccharomyces cerevisiae, a mutation in the universally conserved hinge region of the trigger loop, E1103G, induces a gain of function in the Pol II elongation rate, whereas the corresponding mutation in Pol I, E1224G, results in a loss of function. The E1103G Pol II mutation stabilizes the closed conformation of the trigger loop, promoting the catalytic step, the putative rate-limiting step for Pol II. In single-nucleotide and multinucleotide addition assays, we observe a decrease in the rate of nucleotide addition and dinucleotide cleavage activity by E1224G Pol I and an increase in the rate of misincorporation. Collectively, these data suggest that Pol I is at least in part rate-limited by the same step as Pol II, the catalytic step.