TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE FROM ESCHERICHIA-COLI - RECOGNITION OF FULL-LENGTH QUEUINE-COGNATE TRANSFER-RNAS

A key enzyme involved in the incorporation of the modified base queuin e into tRNA (position 34) is tRNA-guanine transglycosylase (TGT), Stud ies of the recognition of truncated tRNAs by the Escherichia coli TGT have established a minimal recognition motif involving a minihelix wit h a 7 base loop containing a U-(G) under bar-U sequence (where G is re placed with queuine) [Curnow, A.W. and Garcia, G.A. (1995) J. Biol. Ch em. 270, 17264-17267; Nakanishi, S, et al, (1994) J, Biol, Chem, 269, 32221-32225], Still, a clearer understanding of the recognition of ful l-length 'queuine-cognate' tRNAs by TGT remains lacking. In this paper , me report the in vitro transcription and enzymological characterizat ion (K-m and k(cat)) of all four 'queuine-cognate' tRNAs from E. coli and from Saccharomyces cerevisiae with the TGT from E, coli, No primar y or secondary structures emerge as important recognition elements fro m this study. The modest differences in substrate specificity (relativ e k(cat)/K-m values vary from 0.5 to 8.4) seen among these 'queuine-co gnate' tRNAs most likely result from the accumulated effects of many s ubtle factors. Interestingly, the yeast tRNAs are essentially equivale nt to the E, coli tRNAs as substrates for TGT, indicating that there i s nothing intrinsic to the yeast tRNAs that accounts for the absence o f queuine in yeast. (C) 1998 Federation of European Biochemical Societ ies.