Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27.
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.
Matsumoto
T
T
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.
Nishikawa
K
K
Hori
H
H
Ohta
T
T
Miura
K
K
Watanabe
K
K
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002175:Candida / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013824:Thermus / Q000201:enzymology / Q000235:genetics*; D012359:tRNA Methyltransferases / Q000235:genetics*
D000596:Amino Acids; D012358:RNA, Transfer, Met; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase; D012260:Ribonucleases