Ramesh V, RajBhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2001 Feb 2;276(5):3660-5. doi: 10.1074/jbc.M008206200. Epub 2000 Oct 31.
The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.
在古细菌中，氨酰 - tRNA合成酶和翻译因子识别tRNA的模式在很大程度上尚不清楚。为了研究这个过程，我们已将来自中度嗜盐古细菌嗜盐栖热菌的野生型起始tRNA基因及其衍生的突变体克隆到一个能够在这些细胞中表达tRNA的质粒中。对体内tRNA的分析表明，起始tRNA在嗜盐栖热菌中被氨酰化，但未被甲酰化。这一结果为古细菌中蛋白质合成起始于甲硫氨酸而非甲酰甲硫氨酸这一观点提供了直接支持。我们分析了起始tRNA反密码子序列中的两种不同突变（CAU→CUA和CAU→GAC）对其在体内被氨酰 - tRNA合成酶识别的影响。CAU→CUA突变体在体内未被显著氨酰化，这表明反密码子在甲硫氨酰 - tRNA合成酶对tRNA进行氨酰化过程中具有重要作用。然而，这种突变的起始tRNA在体外可被大肠杆菌谷氨酰胺 - tRNA合成酶氨酰化，这表明缺乏氨酰化是由于嗜盐栖热菌中缺乏能够识别该突变tRNA的合成酶。古细菌缺乏谷氨酰胺 - tRNA合成酶，并利用一种涉及谷氨酰胺 - tRNA合成酶和谷氨酰胺酰胺转移酶的两步途径来生成谷氨酰胺 - tRNA。突变tRNA缺乏氨酰化表明该突变tRNA不是嗜盐栖热菌谷氨酰胺 - tRNA合成酶的底物。CAU→GAC反密码子突变体在体内很可能被缬氨酸氨酰化。因此，反密码子在至少两种嗜盐菌氨酰 - tRNA合成酶识别tRNA的过程中起着重要作用。
