Zhang J R, Deutscher M P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
J Bacteriol. 1988 Feb;170(2):522-7. doi: 10.1128/jb.170.2.522-527.1988.
RNase D is a 3'-exoribonuclease whose in vitro specificity has suggested that it is involved in the processing of tRNA precursors. Its in vivo role has remained unclear, however, because mutant cells devoid of the enzyme display no defect in growth or tRNA processing. To learn more about the structure and function of RNase D, we cloned the Escherichia coli rnd gene, which is thought to code for this enzyme. The rnd gene was isolated from a cosmid library based on elevated RNase D activity and was subcloned as a 1.4-kilobase-pair fragment in pUC18. Maxicell analysis of the cloned fragment revealed that a single protein of approximately 40 kilodaltons, which is the size of RNase D, was synthesized. The rnd gene is present as a single copy on the E. coli chromosome and is totally absent in a deletion mutant. Cells that harbored the cloned rnd gene displayed RNase D activity that was elevated as much as 20-fold over that of the wild type. As growth of the culture progressed, however, RNase D specific activity declined dramatically, together with a similar decrease in plasmid copy number. In contrast, no decrease in copy number was observed with an inactive rnd gene. Placement of the rnd gene downstream from the lac promoter led to inducible RNase D overexpression and concomitantly slowed cell growth. These findings support the idea that rnd is the structural gene for RNase D and indicate that elevated RNase D activity is deleterious to E. coli.
核糖核酸酶D是一种3'-外切核糖核酸酶，其体外特异性表明它参与了转运RNA前体的加工过程。然而，其在体内的作用仍不清楚，因为缺乏该酶的突变细胞在生长或转运RNA加工方面没有缺陷。为了更多地了解核糖核酸酶D的结构和功能，我们克隆了大肠杆菌的rnd基因，该基因被认为编码这种酶。rnd基因是从一个基于核糖核酸酶D活性升高的黏粒文库中分离出来的，并作为一个1.4千碱基对的片段亚克隆到pUC18中。对克隆片段的最大细胞分析表明，合成了一种大小约为40千道尔顿的单一蛋白质，这正是核糖核酸酶D的大小。rnd基因在大肠杆菌染色体上以单拷贝形式存在，而在一个缺失突变体中则完全不存在。携带克隆的rnd基因的细胞所显示的核糖核酸酶D活性比野生型高出多达20倍。然而，随着培养物的生长，核糖核酸酶D的比活性急剧下降，同时质粒拷贝数也有类似的减少。相比之下，对于一个无活性的rnd基因，未观察到拷贝数的减少。将rnd基因置于乳糖启动子下游导致了可诱导的核糖核酸酶D过表达，并随之减缓了细胞生长。这些发现支持了rnd是核糖核酸酶D的结构基因这一观点，并表明核糖核酸酶D活性升高对大肠杆菌是有害的。
