Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27

Abstract

Recognition sites of tRNA by tRNA(guanosine-2'-)-methyltransferase (Gm-methylase) [EC 2.1.1.34] from an extreme thermophile, Thermus thermophilus HB27, were studied by two independent methods--fragment reactions and footprinting analyses, using yeast tRNA(Phe) and Escherichia coli tRNA(fMet) as substrates. None of the tRNA-derived oligonucleotides which have the G-G sequence but are not long enough to form the "stem-loop" structure could be methylated by Gm-methylase. The 5'-half fragments having the intact D-"stem-loop" structure served as substrates for Gm-methylase, with a similar Vmax but 6-8 times larger Km, as compared with the intact tRNAs. The results of footprinting analyses were consistent with the foregoing findings. Gm-methylase protected only the D-loop region of tRNA from RNase T1 attack, but other parts of tRNA extending from the amino acid stem to the T arm became more sensitive to RNase T1, suggesting a considerable change of tRNA tertiary structure due to complex formation with Gm-methylase. These results indicate that a D-"stem-loop" structure is a prerequisite for recognition by Gm-methylase.