PMID:11705403
| Citation |
Steiger, MA, Kierzek, R, Turner, DH and Phizicky, EM (2001) Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog. Biochemistry 40:14098-105 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Kinetics; NAD/metabolism; Oligodeoxyribonucleotides/metabolism; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)/biosynthesis; Phosphotransferases (Alcohol Group Acceptor)/genetics; Phosphotransferases (Alcohol Group Acceptor)/isolation & purification; Phosphotransferases (Alcohol Group Acceptor)/metabolism; RNA Splicing; RNA, Bacterial/metabolism; RNA, Fungal/metabolism; RNA, Transfer/genetics; RNA, Transfer/metabolism; RNA, Transfer, Phe/metabolism; RNA, Transfer, Tyr/metabolism; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/isolation & purification; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae Proteins; Substrate Specificity/genetics |
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