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 Sep;312:107281. doi: 10.1016/j.bpc.2024.107281. Epub 2024 Jun 8.
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.
RNA 聚合酶 I（Pol I）负责合成核糖体 RNA，这是核糖体生物发生中的限速步骤。我们已经报道了 Pol I 催化的连续核苷酸添加过程中限速步骤的速率常数的幅度存在广泛的可变性。在这项研究中，我们试图确定碱基身份是否会影响 Pol I 催化的核苷酸添加的限速步骤。为此，我们报告了对 Pol I 催化的 AMP、CMP、GMP 和 UMP 掺入的瞬态动力学研究。我们发现 Pol I 使用一种动力学机制来掺入所有核苷酸。然而，我们发现 UMP 的掺入速度比 AMP、CMP 和 GMP 的添加速度快。此外，我们发现当 3'末端碱基是 UMP 时，从 3'端内切切除二聚体最快。先前已经表明，下游和上游模板序列的身份都会影响核苷酸添加的动力学。这里报道的结果表明，进入碱基的身份也会影响观察到的限速步骤的幅度。
