Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I
Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I
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DOI：
10.1016/j.bpj.2021.09.008
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发表时间：
2021-10-19
影响因子：
3.4
通讯作者：
Lucius, Aaron L.
中科院分区：
文献类型：
--
作者：
Ingram, Zachariah M.;Schneider, David A.;Lucius, Aaron L.
RNA聚合酶执行基因表达的第一步：将DNA转录成RNA。与原核生物不同，真核生物至少表达三种特殊的核多亚基RNA聚合酶（Pol I， Pol II和Pol III）。RNA聚合酶I （Pol I）合成最丰富的RNA——核糖体RNA。近60%的总转录致力于核糖体RNA合成，使其成为细胞最消耗能量的任务之一。虽然已经报道了Pol I催化核苷酸加成的动力学机制，但尚不清楚不同核苷酸序列对掺入速率常数的影响程度。此外，目前尚不清楚先前对单核苷酸结合的研究是否对易位步骤敏感。在这里，我们发现Pol I在k（max）和k -1/2值上都表现出相当大的变异性，使用体外多ntp掺入实验测量AMP和GMP掺入。我们发现前两个观察到的核苷酸结合表现出更快的k（max）值（类似于200秒（-1）），而其余七个位置（类似于60秒（-1））。此外，与GTP相比，Pol I与GTP结合的平均K-1/2大约是GTP的三倍，这表明Pol I与GTP的亲和力比ATP更强。我们的结果表明，Pol I在描述每个核苷酸结合的观察速率常数中表现出显著的可变性。了解Pol酶之间的差异将有助于了解导致其特殊作用的进化压力。因此，这项工作的结果对于与所有生命领域的其他聚合酶进行比较至关重要。
RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell's most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Furthermore, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here, we show that Pol I exhibits considerable variability in both k(max) and K-1/2 values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster k(max)-values (similar to 200 s(-1)) compared with the remaining seven positions (similar to 60 s(-1)). Additionally, the average K-1/2 for ATP incorporation was found to be approximately threefold higher compared with GTP, suggesting Pol I has a tighter affinity for GTP compared with ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.