Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada.
Sci Rep. 2012;2:663. doi: 10.1038/srep00663. Epub 2012 Sep 17.
Cbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5's affinity for tRNA, but they also directly enhance Cbf5's catalytic activity by increasing the k(cat) of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.
Cbf5 是一种假尿嘧啶核苷合成酶，通常作为 H/ACA 小核糖核蛋白的一部分，以指导 RNA 依赖的方式发挥作用；然而，古菌 Cbf5 也可以在不依赖指导 RNA 的情况下修饰 tRNA 中的尿嘧啶 55。Cbf5 的这种非指导 RNA 活性可以通过在 H/ACA 小核糖核蛋白中也发现的 Nop10 和 Gar1 蛋白增强。在这里，我们分析了 Nop10 和 Gar1 对 Cbf5 催化 tRNA 假尿嘧啶化的特定贡献。有趣的是，Nop10 和 Gar1 不仅增加了 Cbf5 对 tRNA 的亲和力，而且通过增加反应的 k(cat)直接增强了 Cbf5 的催化活性。与依赖指导 RNA 的反应不同，Gar1 不参与 tRNA 修饰后的产物释放。这些结果连同结构信息表明，Nop10 和 Gar1 将 Cbf5 稳定在其活性构象中；我们假设这对于 Cbf5 依赖指导 RNA 的假尿嘧啶形成也是如此。
