Mode of action of RNase BN/RNase Z on tRNA precursors: RNase BN does not remove the CCA sequence from tRNA.
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.
Dutta
Tanmay
T
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33101, USA.
Deutscher
Murray P
MP
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004722:Endoribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D008670:Metals; D012322:RNA Precursors; D012343:RNA, Transfer; D004722:Endoribonucleases; D005095:Exoribonucleases; C110250:RNase Z; C038206:ribonuclease BN