Major identity element of glutamine tRNAs from Bacillus subtilis and Escherichia coli in the reaction with B. subtilis glutamyl-tRNA synthetase

Abstract

Early investigations revealed that Bacillus subtilis glutamyl-tRNA synthetase [GluRS (bs)] is responsible for aminoacylating both glutamate tRNA [tRNA(Glu) (bs)] and glutamine tRNA [tRNA(Gln) (bs)] with glutamate. The same Bacillus enzyme can also efficiently attach glutamate to one isoacceptor glutamine tRNA [tRNA(Gln) (ec)] of Escherichia coli in vitro but not to tRNA2(Gln) (ec) and tRNA(Glu) (ec). To characterize identity elements of these glutamine tRNAs in the interaction with GluRS (bs), tRNA2(Gln) (ec), tRNA1(Gln) (ec), three other mutant glutamine tRNAs [tRNA2(Gln) (AU) (C34 --> U34), tRNA2(Gln) (12M) (C34 --> U34, 31A-U39 --> 31U-A39), and tRNA2(Gln) (M21) (64C --> G50 --> 64G-C50, 63U-A51 --> 63A-U51)] originated from tRNA2(Gln) (ec), tRNA(Gln) (bs), and a mutant tRNAM(Gln) (bs) whose U at the 34th position (U34), was replaced to C (C34), were produced in E. coli. All of the E. coli glutamine tRNAs containing U34 such as tRNA1(Gln), tRNA2(Gln) (AU), and tRNA2(Gln) (12M) could be charged with glutamate by GluRS (bs), whereas tRNA2(Gln) (ec) and its T-stem mutant tRNA2(Gln) (M21) containing C34 could not be charged by the same enzyme. The unique change of C34 to U34 of tRNA2(Gln) (ec) acquired glutamate acceptor activity by GluRS (bs). This result suggests that the U34 is the major identity element of tRNA1(Gln) (ec) in the recognition by GluRS (bs). The same situation was found in tRNA(Gln) (bs). The glutamate acceptor activity of tRNA(Gln) (bs) disappeared on replacement of U34 to C34. To find out whether modified bases in tRNA(Gln) (bs) are involved in the recognition by GluRS (bs), glutamylation of tRNA(Gln) (bs) produced by in vitro transcription was also examined but the in vitro transcript of tRNA(Gln) (bs) could not be charged with glutamic acid by GluRS (bs). All of these mean that the major recognition element for GluRS (bs) is U at the 34th position of both tRNA(Gln) (bs) and tRNA1(Gln) (ec) as a modified form.