Mode of Action of RNase BN/RNase Z on tRNA Precursors: RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA

Tanmay Dutta,Murray P Deutscher

doi:10.1074/jbc.m110.141101

Abstract

RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3'-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087-1097). We show that purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co(2+) resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg(2+), RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.

Highlights

3Ј-Maturation of tRNA precursors differs among organisms depending on whether or not the universal 3Ј-terminal CCA sequence is encoded [1,2,3,4,5,6]
RNase BN was shown to be required for maturation of certain phage T4 tRNA precursors that lack an encoded CCA sequence [11, 22], and such an activity is consistent with that of other RNase Z enzymes, which generally cleave after the discriminator residue [6, 18]
We have reexamined the action of RNase BN on representative E. coli tRNA precursors that contain a CCA sequence and on a phage T4 precursor and a B. subtilis precursor that lack the CCA sequence to ascertain the products of RNase BN action and to determine whether RNase BN functions as an exoribonuclease or endoribonuclease on these substrates

Summary

RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA*

3Ј-Maturation of tRNA precursors differs among organisms depending on whether or not the universal 3Ј-terminal CCA sequence is encoded [1,2,3,4,5,6] In eukaryotic cells, this sequence is absent from tRNA precursors, and the 3Ј-processing step is catalyzed by the endoribonuclease, RNase Z or 3Ј-tRNase, which cleaves at the discriminator base to allow subsequent CCA addition by tRNA nucleotidyltransferase [3, 5, 7]. Mode of Action of RNase BN sors are extremely poor substrates for RNase BN under any condition tested, which may explain why RNase BN is so poor at total tRNA maturation in vivo These findings provide important information on the catalytic capabilities of RNase BN on tRNA precursors and help explain the known physiological properties of this enzyme

EXPERIMENTAL PROCEDURES

RESULTS

Treatment of this precursor also generated a product that

DISCUSSION