E. coli glutamyl-tRNA synthetase is inhibited by anticodon stem-loop domains and a minihelix

Portions of E. coli tRNA(Glu) having identity determinants for glutamyl-tRNA synthetase (ERS, EC 6.1.1.17) have been designed to be the first RNA inhibitors of a Class I synthetase. ERS recognizes the 2-thionyl group of 2-thio-5-methylaminomethyluridine (mnm(5)s(2)U(34)) in the first or wobble anticodon position of E. coli tRNA(Glu). The interaction, as revealed by structural analysis, though specific, appears tenuous. Thus, it is surprising that RNAs designed from this tRNA's anticodon stem and loop domain with (ASL(Glu)-s(2)U(34)) and without s(2)U(34) are bound by ERS and inhibit aminoacylation of the native tRNA. ASL(Glu), ASL(Glu)-s(2)U(34), and a minihelix(Glu) composed of identity determinants of the amino acid accepting stem were thermally stable under conditions of aminoacylation (T(m)s = 75 +/- 1.5, 76 +/- 0.9 and 83 +/- 2.0 degrees C, respectively). In binding competition, the modified ASL(Glu)-s(2)U(34) bound ERS with a higher affinity (half maximal inhibiting concentration, IC(50), 5.1 +/- 0.4 microM) than its unmodified counterpart, ASL(Glu) (IC(50), 10.3 +/- 0.6 microM). The minihelix(Glu), ASL(Glu)-s(2)U(34) and ASL(Glu) bound ERS with K(d)s of 9.9 +/- 3.3, 6.5 +/- 1.7 and 20.5 +/- 3.8 microM. ERS aminoacylation of tRNA(Glu) was inhibited by the tRNA fragments. Unmodified ASL(Glu), minihelix(Glu), and ASL(Glu)-s(2)U(34) exhibited a K(ic) of 1.9 +/- 0.2 microM, 4.1 +/- 0.2 microM, and 6.5 +/- 0.1 microM, respectively. The modified ASL(Glu)-s(2)U(34), though having a higher affinity for ERS, may be released more readily and thus, not be as good an inhibitor as the unmodified ASL. Thus, the RNA constructs are effective tools to study RNA-protein interaction.