Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog.
The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.
Steiger
M A
MA
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
Kierzek
R
R
Turner
D H
DH
Phizicky
E M
EM
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D012343:RNA, Transfer; C117348:KptA protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); C106173:TPT1 protein, S cerevisiae