Komine Y, Kitabatake M, Yokogawa T, Nishikawa K, Inokuchi H
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9223-7. doi: 10.1073/pnas.91.20.9223.
We have determined that 10Sa RNA (one of the small stable RNAs found in Escherichia coli) has an interesting structural feature: the 5' end and the 3' end of 10Sa RNA can be arranged in a structure that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10Sa RNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding 10Sa RNA (ssrA) caused a reduction in the rate of cell growth, which was especially apparent at 45 degrees C, and a reduction in motility on semisolid agar. These phenotypic characteristics of the deletion strain (delta ssrA) allowed us to investigate the effects of some mutations in 10Sa RNA in vivo, although the exact function of 10Sa RNA still remains unclear. When the G.U pair (G3.U357) in 10Sa RNA, which may be equivalent to the determinant G.U pair of alanine tRNA, was changed to a G.A or G.C pair, the ability to complement the phenotypic mutations of the delta ssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a G.A pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (alaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of 10Sa RNA are indeed manifested in vivo.
我们已经确定,10Sa RNA(在大肠杆菌中发现的一种小稳定RNA)具有一个有趣的结构特征:10Sa RNA的5'端和3'端可以排列成一种结构,该结构等同于大肠杆菌丙氨酸tRNA的半分子(受体茎和TFC茎环)。对从具有温度敏感性RNase P功能的细菌突变体中提取的10Sa RNA进行引物延伸分析表明,10Sa RNA的前体(pre-10Sa RNA)在体内折叠成类似前体tRNA的结构,从而可以被RNase P切割以产生成熟10Sa RNA的5'端。纯化的10Sa RNA在体外可以携带丙氨酸。编码10Sa RNA(ssrA)的基因的破坏导致细胞生长速率降低,这在45摄氏度时尤为明显,并且在半固体琼脂上的运动性降低。缺失菌株(ΔssrA)的这些表型特征使我们能够在体内研究10Sa RNA中一些突变的影响,尽管10Sa RNA的确切功能仍不清楚。当10Sa RNA中可能等同于丙氨酸tRNA的决定簇G·U对(G3·U357)变为G·A或G·C对时,互补ΔssrA菌株表型突变的能力丧失。此外,由决定簇碱基被G·A对取代引起的无法互补突变表型的情况可以通过在多拷贝质粒上引入编码丙氨酰-tRNA合成酶(alaS)的基因来克服。证据表明,所提出的10Sa RNA的结构特征确实在体内表现出来。