Bowden Katherine E, Wiese Nicholas S, Perwez Tariq, Mohanty Bijoy K, Kushner Sidney R
Department of Genetics, University of Georgia, Athens, Georgia, USA.
Department of Microbiology University of Georgia, Athens, Georgia, USA.
J Bacteriol. 2017 Oct 17;199(22). doi: 10.1128/JB.00301-17. Print 2017 Nov 15.
RNase PH, encoded by the gene, is a 3'→5' exoribonuclease that in participates primarily in the 3' maturation of pre-tRNAs and the degradation of rRNA in stationary-phase cells. Interestingly, the routinely used laboratory strains of MG1655 and W3110 have naturally acquired the allele, encoding a truncated catalytically inactive RNase PH protein which is widely assumed to be benign. Contrary to this assumption, we show that the -encoded Rph-1 protein inhibits RNase P-mediated 5'-end maturation of primary pre-tRNAs with leaders of <5 nucleotides in the absence of RppH, an RNA pyrophosphohydrolase. In contrast, RppH is not required for 5'-end maturation of endonucleolytically generated pre-tRNAs in the strain and for any tRNAs in Δ mutant or strains. We propose that the Rph-1 protein bound to the 3' end of the substrate creates a steric hindrance that in the presence of a triphosphate at the 5' end reduces the ability of RNase P to bind to the pre-tRNA. In this paper, we demonstrate that the mutation found in commonly used strains leads to the synthesis of a truncated functionally inactive RNase PH protein that interferes with the 5'-end maturation of specific tRNAs with short 5' leaders by RNase P in the absence of RppH, an RNA pyrophosphohydrolase that converts primary 5' triphosphates into 5' monophosphates. The data presented indicate that the presence of the triphosphate interferes with RNase P binding to the pre-tRNA.
由该基因编码的核糖核酸酶PH（RNase PH）是一种3'→5'外切核糖核酸酶，在大肠杆菌中主要参与前体tRNA的3'端成熟以及稳定期细胞中rRNA的降解。有趣的是，常用的实验室菌株MG1655和W3110已自然获得该等位基因，其编码一种截短的无催化活性的RNase PH蛋白，人们普遍认为该蛋白是无害的。与这一假设相反，我们发现，在没有RNA焦磷酸水解酶RppH的情况下，该等位基因编码的Rph-1蛋白会抑制RNase P介导的、前导序列小于5个核苷酸的初级前体tRNA的5'端成熟。相比之下，在Δrph-1菌株中，内切核酸酶产生的前体tRNA的5'端成熟以及在ΔrppH突变体或Δrph-1ΔrppH菌株中的任何tRNA的5'端成熟都不需要RppH。我们提出，与底物3'端结合的Rph-1蛋白会产生空间位阻，在5'端存在三磷酸的情况下，会降低RNase P与前体tRNA结合的能力。在本文中，我们证明，在常用的大肠杆菌菌株中发现的该突变会导致截短的、无功能活性的RNase PH蛋白的合成，该蛋白在没有将初级5'三磷酸转化为5'单磷酸的RNA焦磷酸水解酶RppH的情况下，会干扰RNase P对具有短5'前导序列的特定tRNA的5'端成熟。所呈现的数据表明，三磷酸的存在会干扰RNase P与前体tRNA的结合。
