Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1 Journal Article
| Authors: | Jain, R.; Shuman, S. |
| Article 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 horikoshiiClp1 (PhoClp1) reveals it to be a thermostable 5'-OH polynucleotide kinase optimally active at 55°Cto85°C. PhoClp1 catalyzes transfer of the gamma phosphate from ATP (K<sub>m</sub> 16 μM) 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. Copyright © 2009 RNA Society. |
| Keywords: | protein expression; unclassified drug; gene mutation; nonhuman; protein function; protein analysis; enzyme activity; transcription factors; nuclear proteins; rna; dna; amino acid sequence; molecular sequence data; messenger rna; protein purification; eukaryota; recombinant protein; protein transport; temperature; saccharomycetales; alanine; catalysis; adenosine triphosphate; sequence homology; transfer rna; oligonucleotide; phosphate; phosphotransferase; sodium chloride; aspartic acid; genetic conservation; rna end healing; trna splicing; clp1 enzyme; endopeptidase clp; lysine; polynucleotide 5' hydroxyl kinase; archaebacterium; protein cleavage; pyrococcus horikoshii; thermostability; phosphotransferases; polynucleotide 5'-hydroxyl-kinase; rna ligase (atp); archaea; pyrococcus |
| Journal Title: | RNA |
| Volume: | 15 |
| Issue: | 5 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2009-05-01 |
| Start Page: | 923 |
| End Page: | 931 |
| Language: | English |
| DOI: | 10.1261/rna.1492809 |
| PUBMED: | 19299550 |
| PROVIDER: | scopus |
| PMCID: | PMC2673061 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 2" - "Export Date: 30 November 2010" - "CODEN: RNARF" - "Source: Scopus" |
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