PURIFICATION AND CHARACTERIZATION OF A TRANSFER-RNA NUCLEOTIDYLTRANSFERASE FROM LUPINUS-ALBUS AND FUNCTIONAL COMPLEMENTATION OF A YEAST MUTATION BY THE CORRESPONDING CDNA

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to ap parent homogeneity from a crude extract of Lupinus albus seeds. Purifi cation was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylap atite and affinity columns. The lupin enzyme exhibited a pH optimum an d salt and ion requirements that were similar to those of tRNA nucleot idyltransferases from other sources. Oligonucleotides, based on partia l amino acid sequence of the purified protein, were used to isolate th e corresponding cDNA. The cDNA potentially encodes a protein of 560 am ino acids with a predicted molecular mass of 64164 Da in good agreemen t with the apparent molecular mass of the pure protein determined by s odium 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 al three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitiv e mutation in the yeast tRNA nucleotidyltransferase gene. While the lu pin cDNA compensated for the nucleocytoplasmic defect in the yeast mut ant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.