Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by corresponding cDNA.
ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.
Shanmugam
K
K
Department of Chemistry, Concordia University, Montreal, Quebec, Canada.
Hanic-Joyce
P J
PJ
Joyce
P B
PB
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000201:enzymology; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D015723:Gene Library; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase