Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Department of Chemistry, McGill University, Montreal, Quebec, Canada H3A0B8.
RNA. 2019 Jul;25(7):783-792. doi: 10.1261/rna.071142.119. Epub 2019 Apr 24.
The enzyme Tpt1 removes the 2'-PO at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2'-PO attacks NAD to form an RNA-2'-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2'-PO removal, via their unique NAD-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD-dependent conversion of RNA terminal 2' and 3' monophosphate ends to 2'-OH and 3'-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. and Tpt1 proteins are active on 2'-PO and 3'-PO ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO The accumulation of a terminal 3'-phospho-ADP-ribosylated RNA intermediate during the 3'-phosphotransferase reaction suggests that the geometry of the 3'-p-ADPR adduct is not optimal for the ensuing transesterification step. Tpt1 acts specifically on a terminal 2'-PO end and not with a 3'-PO In contrast, Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO or 3'-PO end.
酶 Tpt1 去除真菌 tRNA 连接酶产生的剪接连接处的 2'-PO；它通过两步反应实现，其中（i）内部 RNA 2'-PO 攻击 NAD 以形成 RNA-2'-磷酸-ADP-核糖基中间物；（ii）核糖 O2″到 2'-磷酸二酯的酯交换产生 2'-OH RNA 和 ADP-核糖-1″,2″-环磷酸产物。Tpt1 酶在没有真菌型 RNA 连接酶的分类群中所起的作用仍然不清楚。一个有吸引力的前景是，Tpt1 酶可能通过其独特的 NAD 依赖性转移酶机制催化除内部 RNA 2'-PO 去除以外的反应。本研究扩展了 Tpt1 酶家族的功能范围，包括 NAD 依赖性转化 RNA 末端 2'和 3'单磷酸末端为 2'-OH 和 3'-OH 末端。显著的发现是不同的 Tpt1 酶在末端磷酸去除的能力和位置特异性上有所不同。和 Tpt1 蛋白对 2'-PO 和 3'-PO 末端具有活性，对 2'-PO 的动力学偏好为 2.4-2.6 倍。在 3'-磷酸转移酶反应中积累末端 3'-磷酸-ADP-核糖基化 RNA 中间物表明 3'-p-ADPR 加合物的几何形状对于随后的酯交换步骤不是最佳的。Tpt1 特异性作用于末端 2'-PO 末端，而不是 3'-PO 末端。相比之下，和 Tpt1 不能有效去除 2'-PO 或 3'-PO 末端。
