Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites.
We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.
Belford
H G
HG
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
Westaway
S K
SK
Abelson
J
J
Greer
C L
CL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D011487:Protein Conformation; D011130:RNA Ligase (ATP) / Q000378:metabolism* / Q000648:ultrastructure; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D011130:RNA Ligase (ATP)