Bailly Marc, Giannouli Stamatina, Blaise Mickael, Stathopoulos Constantinos, Kern Daniel, Becker Hubert Dominique
Département Machineries traductionnelles, UPR 9002 Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS 15, Rue René Descartes, F-67084 Strasbourg Cédex, France.
Nucleic Acids Res. 2006;34(21):6083-94. doi: 10.1093/nar/gkl622. Epub 2006 Oct 29.
In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1-A72 whereas the G1-C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.
在许多原核生物和细胞器中，天冬酰胺和谷氨酰胺由一种依赖tRNA的酰胺转移酶（AdT）形成，该酶分别催化错误负载在tRNAAsn和tRNAGln上的天冬氨酸和谷氨酸的酰胺化反应。这些途径弥补了生物体中天冬酰胺基-tRNA合成酶和谷氨酰胺基-tRNA合成酶的不足，并为翻译机制提供Asn-tRNAAsn和Gln-tRNAGln。到目前为止，关于赋予tRNA在同源氨基酸形成中特定辅助因子作用的结构元件还一无所知。我们在此表明，使用天冬酰胺化的tRNAAsn和tRNAAsp变体，U1-A72碱基对促进了酰化tRNAAsn中天冬氨酸的酰胺化，而G1-C72碱基对以及tRNAAsp的D环中额外核苷酸U20A的存在则阻止了酰胺化。基于对使用依赖AdT途径形成Gln-tRNAGln的细菌的tRNAGln和tRNAGlu序列比对的比较，我们预测相同的核苷酸组合也决定了特定的依赖tRNA的Gln形成。相反，我们表明古细菌AdT将天冬氨酸依赖tRNA转化为天冬酰胺主要由可变区的核苷酸G46和U47介导。根据这些结果，我们提出细菌和古细菌AdT利用特定于王国的信号来催化依赖tRNA的天冬酰胺和谷氨酰胺的形成。
