The peculiar architectural framework of tRNA^Sec is fully recognized by yeast AspRS

Abstract

The wild-type transcript of Escherichia coli tRNA^Sec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNA^Sec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNA^Asp and is accounted for by the presence of the discriminator residue G₇₃, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G₃₄, U₃₅, C₃₆, C₃₈) into tRNA^Sec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNA^Asp. Expression of the aspartate identity set in tRNA^Sec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNA^Sec and tRNA^Asp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.