tRNA-guanine Transglycosylase from Escherichia coli

Abstract

Previously, we have demonstrated that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is capable of utilizing an in vitro generated 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 tRNA structural motifs necessary for recognition comprise a loop at the end of a short helix. To gain further insight into the structural requirements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analyses at 20 and 37 degrees C indicate that this minihelix is predominantly melted at 37 degrees C and that the melted conformation is not a substrate for TGT. This is confirmed by the determination that a non-helical analogue of the minihelix is not a substrate for TGT at either temperature. Two additional minihelices designed to be stable at 37 degrees C, ECYMH (a 4-base pair extension of the previous minihelix) and SCDMH (a yeast tRNA(Asp) analogue of ECYMH), were generated and characterized. Finally, several sequence mutants of SCDMH, focusing on the G30U40 base pair and U33G34U35 loop sequence, have been produced, and kinetic parameter determinations have been performed at 37 degrees 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.