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_1/2values 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 (∼200 s⁻¹) compared with the remaining seven positions (∼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.

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化的核多亚基 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，即核糖体 RNA。近 60% 的总转录用于核糖体 RNA 合成，使其成为细胞最耗能的任务之一。虽然已经报道了由 Pol I 催化的核苷酸添加的动力学机制，但仍不清楚不同的核苷酸序列在多大程度上影响掺入率常数。此外，目前尚不清楚先前对单核苷酸掺入的研究是否对易位步骤敏感。在这里，我们表明 Pol I 在两个 k 中都表现出相当大的可变性使用体外多 NTP 掺入测定法测量 AMP 和 GMP 掺入的_最大和 K _1/2值。我们发现前两个观察到的核苷酸掺入表现出更快的 k_最大值（~200 s ^-1）与其余七个位置（~60 s ^-1）相比。此外，平均 K _1/2与 GTP 相比，发现 ATP 掺入大约高出三倍，这表明与 ATP 相比，Pol I 对 GTP 的亲和力更紧密。我们的结果表明，Pol I 在描述每个核苷酸掺入的观察到的速率常数中表现出显着的可变性。了解 Pol 酶之间的差异将有助于深入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。