Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, AL 35294, USA.
Biophys Chem. 2024 Nov;314:107302. doi: 10.1016/j.bpc.2024.107302. Epub 2024 Aug 3.
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) 来进行转录，而细菌和古菌只使用一种。我们使用瞬态动力学方法，广泛研究和比较了三种 Pols 催化的单核苷酸和多核苷酸添加的动力学。在单核苷酸添加实验中，我们观察到了超过一个掺入的意外延伸产物，这可以归因于错配掺入、几乎无法检测到的污染 NTPs 的存在，或者两者的混合物。在这里，我们报告了一种分析策略的开发和验证，用于在出现这种情况时解释意外延伸产物的存在。使用这种方法，我们发现了证据表明，非互补核苷酸在ΔA12 Pol I 和 Pol II 的延伸复合物的活性位点与互补核苷酸在热力学上竞争。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，而竞争性结合表明互补和非互补核苷酸的亲和力在数量级内。因此，我们表明，应用我们的方法将允许提取报告核苷酸进入和选择性的能量学的其他信息。
