RNA-editing terminal uridylyl transferase 1: identification of functional domains by mutational analysis.
The catalytic, RNA-binding and oligomerization domains of the RNA-editing terminal uridylyl transferase 1 (RET1) from Leishmania tarentolae mitochondria were characterized by mutational analysis. Significant N- and C-terminal portions of the protein were found to be dispensable for UTP polymerization in vitro. Changes of conserved amino acids in the active site demonstrated a general similarity of sugar-phosphate moiety recognition of the incoming ribonucleotide triphosphate by RET1 and eukaryotic poly(A) polymerases. Overlapping RNA-binding and oligomerization regions were mapped to the C-terminal region, which is conserved only among trypanosomatid RET1 enzymes. In the absence of an RNA primer, RET1 can use UTP itself to initiate nucleotide transfer and produce poly(U) molecules of several hundred nucleotides. An N-terminal zinc finger motif is essential for enzyme activity; deletion of this motif or chelation of zinc inhibits activity.
Aphasizheva
Inna
I
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles 90095, USA.
Aphasizhev
Ruslan
R
Simpson
Larry
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D002417:Cattle; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D017353:Gene Deletion; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014544:Uridine Triphosphate / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D000596:Amino Acids; D002241:Carbohydrates; D017931:DNA Primers; D011994:Recombinant Proteins; D012313:RNA; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D008274:Magnesium; D015032:Zinc; D014544:Uridine Triphosphate