Abstract
The pyrophosphorolysis of tRNA by yeast CTP-(ATP):tRNA nucleotidyltransferase has been studied in an effort to define the behavior of the enzyme and the experimental parameters that lead to net loss of the 3'-terminal nucleotide or to nucleotide exchange. It was found that removal of AMP from the terminus of tRNA proceeded optimally at 1.0 mM PPi; incorporation of 2'- or 3'-dAMP was also studied and shown to proceed optimally at a 6.0 mM concentration of deoxynucleoside triphosphate. CTP was shown to inhibit the pyrophosphorolysis and nucleotide exchange observed when starting from intact tRNA, but apparently not by inhibiting removal of CMP from tRNA missing the 3'-terminal adenosine moiety. The optimized conditions for nucleotide exchange were used for the preparative conversion of tRNAs to species terminating in 2'- and 3'-deoxyadenosine.
Highlights
The pyrophosphorolysis of tRNAbyyeast CTP(ATP):tRNA nucleotidyltransferase has been studied in an effort to definethe behavior of the enzyme and the experimental parametersthat lead to net loss of the 3’terminal nucleotide or to nucleotide exchange
The time course of radiolabel incorporation into tRNAindicated that theinitial rate of exchange was proportional to added pyrophosphate up to 1.0 mM concentration, the optimal concentration for adenine nucleotide exchange
The function(s) [12,13,14] and substrate specificity [1, 6, 7, 12, 22] of CTP(ATP):tRNA nucleotidyltransferase have been studied extensively and the enzymes from several sources have been shown to effect the pyrophosphorolysis of tRNA [12,23,24,25,26,27,28], there is a paucity of evidence in the literature for concomitantpyrophosphate exchange [24] and no systematicstudy of the forward and reverse reactions has appeared
Summary
In addition to contributing to an understanding of CTP(ATP):tRNA nucleotidyltransferase function, the results should provide access,in asingle step, to a variety of tRNAs modified at the 3’-end. This potential is illustrated by the conversion of Escherichia coli tRNAs [1]to the respective species terminating in 2’- or 3’-deoxyadenosine(2 and 3) in a pyrophosphate-dependent exchange reaction that combines abbreviation and reconstruction into a single reaction
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