Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation

IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biophysical chemistry Pub Date : 2024-06-08 DOI:10.1016/j.bpc.2024.107281
Kaila B. Fuller , Ruth Q. Jacobs , Zachariah I. Carter , Zachary G. Cuny , David A. Schneider , Aaron L. Lucius
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Abstract

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3′ end was fastest when the 3′ terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

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描述 RNA 聚合酶 I 单核苷酸掺入的全局动力学机制揭示了 UMP 的快速掺入
RNA 聚合酶 I(Pol I)负责合成核糖体 RNA,这是核糖体生物发生过程中的限速步骤。在本研究中,我们试图确定碱基特性是否会影响 Pol I 催化的核苷酸加成的限速步骤。为此,我们报告了 Pol I 催化的 AMP、CMP、GMP 和 UMP 加成的瞬态动力学分析。但是,我们发现 UMP 的加入速度快于 AMP、CMP 和 GMP 的加入速度。此外,我们还发现,当 3′末端碱基是 UMP 时,从 3′末端去除二聚体的核酸内切速度最快。以前的研究表明,下游和上游模板序列的同一性都会影响核苷酸添加的动力学。本文报告的结果表明,输入碱基的特征也会影响观察到的速率限制步骤的大小。
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来源期刊
Biophysical chemistry
Biophysical chemistry 生物-生化与分子生物学
CiteScore
6.10
自引率
10.50%
发文量
121
审稿时长
20 days
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.
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