Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
Department of Biochemistry, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Nat Commun. 2021 Oct 8;12(1):5900. doi: 10.1038/s41467-021-26080-7.
RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3'-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3'-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3'-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3'-cleavage activity increases growth rate but is nonessential.
RNA 聚合酶 III 通过终止相关的重新起始-循环利用来实现高水平的 tRNA 合成，其中涉及必需的 C11 亚基和异源二聚体 C37/53。C11-CTD（C 端结构域）促进 Pol III 活性中心内在的 RNA 3'-切割，尽管这种活性的破译功能已经变得复杂。我们表明，C11 的分离 NTD（N 端结构域）刺激 Pol III 由 C37/53 终止，但不刺激重新起始-循环利用，后者需要 NTD-接头（NTD-L）。通过一种不同于当前认为 RNA 3'-切割活性至关重要的方法，我们表明 NTD-L 可以提供酿酒酵母 C11 的必需功能，而经典的阻断切割、干扰活性位点功能且对生长有毒的点突变则不能。生化和体内分析，包括对 C11 不变的中央接头的分析，导致了 Pol III 终止相关的重新起始-循环利用模型。C11 的 NTD 和 CTD 分别刺激终止和 RNA 3'-切割，而 Pol III 特有的重新起始-循环利用活性仅需要 NTD-接头。RNA 3'-切割活性增加生长速率，但不是必需的。
