TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE FROM ESCHERICHIA-COLI - MINIMALTRANSFER-RNA STRUCTURE AND SEQUENCE REQUIREMENTS FOR RECOGNITION

Previously, we have demonstrated that the tRNA-guanine transglycosylas e (TGT) from Escherichia coli is capable of utilizing an in vitro gene rated minihelix consisting of the anticodon stem and loop sequence of E. coli tRNA(Tyr) (Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia , G. A. (1993) Biochemistry 32, 5239-5246). This suggests that the tRN A structural motifs necessary for recognition comprise a loop at the e nd of a short helix. To gain further insight into the structural requi rements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analy ses at 20 and 37 degrees C indicate that this minihelix is predominant ly melted at 37 degrees C and that the melted conformation is not a su bstrate for TGT. This is confirmed by the determination that a non-hel ical analogue of the minihelix is not a substrate for TGT at either te mperature. Two additional minihelices designed to be stable at 37 degr ees C, ECYMH (a 4-base pair extension of the previous minihelix) and S CDMH (a yeast tRNA(Asp) analogue of ECYMH), were generated and char ac terized. Finally, several sequence mutants of SCDMH, focusing on the G (30)U(40) base pair and U(33)G(34)U(35) loop sequence, have been produ ced, and kinetic parameter determinations have been performed at 37 de grees C. Our results are consistent with a recent report (Nakanishi, S ., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K. (1994 ) J. Biol. Chem. 269, 32221-32225) indicating that a UGU sequence in a 7-base loop is the minimal requirement for TGT recognition.