Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases

The position of the tertiary Levitt pair between nucleotides 15 and 48 in t he transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops.. coli tRNA(Gln) possesses the canonical Pu15-Py4 8 trans pairing at this position (G15-C48), while the tRNA(Cys) species fro m this organism instead features an unusual G15-G48 pair. To explore the st ructural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates fo r glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficienci es of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of thi s tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identit y nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tR NA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. S urprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiar y core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G 15-G48 with much better efficiency than those with G15-C48. Therefore, tert iary nucleotides surrounding the Levitt pair must significantly modulate th e efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show t hat the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from sever al groups of G15. The G15-G48 conformations differ significantly compared t o that observed in the native tRNA(Cys) structure bound to EF-Tu, further i mplicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present cruci al recognition determinants to aminoacyl-tRNA synthetases. (C) 2000 Academic Press.