Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions

摘要

In most bacteria and all archaea, glutamyl-tRNA synthetase (GluRS) glutamylates both tRNA~(Glu) and tRNA~(Gln), and then Glu-tRNA~(Gln) is selectively converted to Gln-tRNA~(Gln) by a tRNA-dependent amidotransferase. The mechanisms by which the two enzymes recognize their substrate tRNA(s), and how they cooperate with each other in Gln-tRNA~(Gln) synthesis, remain to be determined. Here we report the formation of the 'glutamine transamidosome' from the bacterium Thermotoga maritima, consisting of tRNA~(Gln), GluRS and the heterotrimeric amidotransferase GatCAB, and its crystal structure at 3.35 A resolution. The anticodon-binding body of GluRS recognizes the common features of tRNA~(Gln) and tRNA~(Glu), whereas the tail body of GatCAB recognizes the outer corner of the L-shaped tRNA~(Gln) in a tRNA~(Gln)-spe-cific manner. GluRS is in the productive form, as its catalytic body binds to the amino-acid-acceptor arm of tRNA~(Gln). In contrast, GatCAB is in the non-productive form: the catalytic body of GatCAB contacts that of GluRS and is located near the acceptor stem of tRNA~(Gln), in an appropriate site to wait for the completion of Glu-tRNA~(Gln) formation by GluRS. We identified the hinges between the catalytic and anticodon-binding bodies of GluRS and between the catalytic and tail bodies of GatCAB, which allow both GluRS and GatCAB to adopt the productive and non-productive forms. The catalytic bodies of the two enzymes compete for the acceptor arm of tRNA~(Gln) and therefore cannot assume their productive forms simultaneously. The transition from the present glutamylation state, with the productive GluRS and the non-productive GatCAB, to the putative amidation state, with the nonproductive GluRS and the productive GatCAB, requires an intermediate state with the two enzymes in their non-productive forms, for steric reasons. The proposed mechanism explains how the transamidosome efficiently performs the two consecutive steps of Gln-tRNA~(Gln) formation, with a low risk of releasing the unstable intermediate Glu-tRNA~(Gln).