Abstract
In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.
Highlights
From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101
We show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity
In Escherichia coli, an organism in which all tRNA genes have the CCA encoded [5], 3Ј maturation generally begins with an endonucleolytic cleavage by RNase E downstream of the mature 3Ј terminus [7], followed by exonucleolytic trimming to expose the CCA sequence [1,2,3,4,5]
Summary
Tion is carried out by an endoribonuclease, termed RNase Z or 3Ј-tRNase [6, 9, 10] Cleavage by this enzyme occurs after the discriminator base [6, 9, 10], followed by CCA addition by tRNA nucleotidyltransferase to generate mature tRNA [5]. The substrate specificity of RNase Z, i.e. removal of incorrect residues in the position of the CCA sequence, was reminiscent of a previously identified E. coli enzyme, termed RNase BN [17] This RNase was shown to be essential for maturation of certain bacteriophage T4 tRNAs that do not encode the CCA sequence [17, 18]; two E. coli mutant strains, BN (a B derivative) and CAN (a K-12 derivative) [19, 20], deficient in this RNase, could not support the growth of a phage T4 mutant that required a suppressor function of phage T4 tRNASer, a tRNA for which the CCA is not encoded [18]. We show here, using biochemical and genetic approaches, that RNase BN is encoded by the elaC gene and, that RNase BN is the RNase Z homologue of E. coli
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