Rampias Theodoros, Sheppard Kelly, Söll Dieter
Department of Molecular Biophysics and Biochemistry and Department of Chemistry, Yale University, New Haven, CT 06511, USA.
Nucleic Acids Res. 2010 Sep;38(17):5774-83. doi: 10.1093/nar/gkq336. Epub 2010 May 10.
Archaea make glutaminyl-tRNA (Gln-tRNA(Gln)) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA(Gln), while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA(Gln). Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA(Asn)) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228-239.]. Here, we show a similar complex for Gln-tRNA(Gln) formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA(Gln) made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K(D) = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA(Gln) formation, with GatDE reducing the affinity of ND-GluRS for tRNA(Glu) by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA(Gln)) formation, and has no major effect on the kinetics of tRNA(Gln) glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.
古菌通过两步过程合成谷氨酰胺基-tRNA（Gln-tRNA(Gln)）；一种非特异性谷氨酰胺-tRNA合成酶（ND-GluRS）形成Glu-tRNA(Gln)，而异二聚体酰胺转移酶GatDE将这种错误负载的tRNA转化为Gln-tRNA(Gln)。许多原核生物以类似的方式合成天冬酰胺基-tRNA（Asn-tRNA(Asn)），使用非特异性天冬酰胺-tRNA合成酶（ND-AspRS）和异三聚体酰胺转移酶GatCAB。转氨体是一种由tRNA合成酶、酰胺转移酶和tRNA组成的复合物，最初是在嗜热栖热菌中针对后一种系统描述的[贝利，M.，布莱斯，M.，洛伯，B.，贝克尔，H.D.和克恩，D.（2007年）转氨体：一种致力于原核生物依赖tRNA的天冬酰胺生物合成的动态核糖核蛋白颗粒。《分子细胞》，28，228 - 239。]。在这里，我们展示了嗜热栖热自养甲烷杆菌中一种类似的用于Gln-tRNA(Gln)形成的复合物，它使得tRNA合成酶产生的错误负载的Glu-tRNA(Gln)能够被引导至酰胺转移酶。古菌ND-GluRS与GatDE的结合（解离常数K(D) = 100 ± 22 nM）隔离了用于Gln-tRNA(Gln)形成的tRNA合成酶，GatDE使ND-GluRS对tRNA(Glu)的亲和力降低了至少13倍。与嗜热栖热菌的转氨体不同，古菌复合物的形成不需要tRNA，通过产物（Gln-tRNA(Gln)）形成时不稳定，并且对tRNA(Gln)的谷氨酰化动力学或转氨作用没有重大影响。这两种转氨体之间的差异可能是由于ND-GluRS是I类氨酰-tRNA合成酶，而ND-AspRS属于II类家族这一事实导致的。
