title: Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1.
abstract: Clp1 proteins are essential components of the eukaryal mRNA 3' cleavage-polyadenylation machinery. Human Clp1 has an additional function as an RNA-specific 5'-OH polynucleotide kinase, which is implicated in RNA end healing. Yeast Clp1 has no kinase activity, although it binds ATP. Here we report that Clp1-like proteins are extant in archaea. Purification and characterization of Pyrococcus horikoshii Clp1 (PhoClp1) reveals it to be a thermostable 5'-OH polynucleotide kinase optimally active at 55 degrees C to 85 degrees C. PhoClp1 catalyzes transfer of the gamma phosphate from ATP (K (m) 16 microM) to either 5'-OH RNA or DNA ends, although it prefers RNA in a competitive situation. Increasing the monovalent salt concentration to 250 mM suppresses the DNA kinase without affecting RNA phosphorylation, suggesting that RNA is a likely substrate for this enzyme in vivo. Indeed, we show that expression of PhoClp1 in budding yeast can complement a lethal mutation in the 5'-OH RNA kinase module of tRNA ligase. PhoClp1 is a member of the P-loop phosphotransferase superfamily. Alanine mutations at the P-loop lysine (Lys49) and a conserved aspartate (Asp73) inactivate the kinase. Our studies fortify emerging evidence for an enzymatic RNA repair capacity in archaea and provide a new reagent for polynucleotide phosphorylation at high temperatures.
authors.affiliation: Graduate Program in Chemical Biology, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Jain
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D012313:RNA / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry
chemical_list: D009687:Nuclear Proteins; D014157:Transcription Factors; D012313:RNA; C520030:CLP1 protein, human; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011130:RNA Ligase (ATP)