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The Purification of 3′ Processing Nucleases Using Synthetic tRNA Precursors

Ranajit K. Ghosh, Murray P. Deutscher


tRNA genes in all cells examined are transcribed initially as large precursor molecules that are converted to the mature species by the successive action of a variety of nucleases (Smith 1976). Two types of tRNA precursors, which differ in the location of the extra residues at the 3′ terminal, have been identified. In one type, which appears to be prevalent in Escherichia coli (Altman and Smith 1971; Chang and Carbon 1975; Schedl et al. 1976), the CCA sequence is already present, and varying numbers of nucleotide residues follow this sequence. Since mutants defective in tRNA nucleotidyl transferase (Deutscher and Hilderman 1974) apparently have little, or no, defect in E, coli tRNA biosynthesis (Morse and Deutscher 1975; Deutscher et al. 1977), the nuclease that removes the extra residues must stop at the CCA sequence, possibly because the tRNA is immediately aminoacylated in vivo. The second type of tRNA precursor, which has been observed in phage-infected E. coli (Barrell et al. 1974) and presumably exists in eukaryotes (Goodman et al. 1977; Valenzuela et al. 1978), contains other nucleotides in place of all or part of the CCA sequence. The nuclease that removes these extra residues must stop at a point that would allow tRNA nucleotidyl transferase to fill in the CCA sequence. From the presumed properties of these putative nuclease activities, it might be expected that at least two different enzymes would be required.

Attempts to identify the enzymes responsible for 3′ trimming of tRNA precursors have led to some confusion. Some...

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