Effects of modification of 4-thiouridine in E. coli tRNA(fMet) on its methyl acceptor activity by thermostable Gm-methylases.
tRNA(guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles, Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D "loop-stem" structure as a minimal requirement. To examine the role of the consensus uridine residue at position 8 (U8) adjacent to the D "loop-stem" region in the recognition of Gm-methylase, 4-thiouridine at this position (s4U8) in Escherichia coli tRNAfMet was modified reversibly with S-benzylthioisothiourea (sBTIU) or irreversibly by UV light. The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with beta-mercaptoethanol. Although both of the modified tRNAfMetS showed larger Km (although to different extents) and slightly smaller Vmax than the intact tRNAfMet, they retained full activities of methylation with tRNA(adenine-1-)-methyltransferase (m1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from T. thermophilus strain HB 27. The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNAfMetS showed methylation efficiency (Vmax/Km) not appreciably different from that of the unmodified 5'-half fragment. These results suggest that the conformation of S4U8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.
Hori
H
H
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo.
Saneyoshi
M
M
Kumagai
I
I
Miura
K
K
Watanabe
K
K
D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism* / Q000528:radiation effects; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism* / Q000528:radiation effects; D013890:Thiourea / Q000031:analogs & derivatives; D013891:Thiouridine / Q000378:metabolism*; D014466:Ultraviolet Rays; D012359:tRNA Methyltransferases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012358:RNA, Transfer, Met; D013891:Thiouridine; C000167:S-benzylthioisothiourea; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase; D013890:Thiourea