Kaila B. Fuller , Ryan M. Requijo , David A. Schneider , Aaron L. Lucius
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引用次数: 0
Abstract
Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two. Here we report the development and validation of an analysis strategy to account for the presence of unexpected extension products, when they occur. Using this approach, we uncovered evidence showing that non-cognate nucleotide, thermodynamically, competes with cognate nucleotide for the active site within the elongation complex of Pol I, ΔA12 Pol I, and Pol II. This observation is unexpected because base pairing interactions provide favorable energetics for selectivity and competitive binding indicates that the affinities of cognate and non-cognate nucleotides are within an order of magnitude. Thus, we show that application of our approach will allow for the extraction of additional information that reports on the energetics of nucleotide entry and selectivity.
真核生物至少表达三种 RNA 聚合酶(Pols)进行转录,而细菌和古细菌只使用一种。利用瞬态动力学,我们对三种 Pols 催化的单核苷酸和多核苷酸添加的动力学进行了广泛的研究和比较。在单核苷酸加成实验中,我们观察到了超出一次加成的意外延伸产物,这可能是由于误加成、存在几乎检测不到的污染 NTP 或两者的混合所致。在此,我们报告了一种分析策略的开发和验证情况,该策略可在出现意外延伸产物时对其进行解释。利用这种方法,我们发现有证据表明,在 Pol I、ΔA12 Pol I 和 Pol II 的延伸复合体中,非识别核苷酸在热力学上与识别核苷酸竞争活性位点。这一观察结果出乎意料,因为碱基配对相互作用为选择性提供了有利的能量,而竞争性结合表明同源核苷酸和非同源核苷酸的亲和力在一个数量级之内。因此,我们的研究表明,应用我们的方法可以提取更多信息,报告核苷酸进入和选择性的能量学。
期刊介绍:
Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.