Catalytic properties of RNase BN/RNase Z from Escherichia coli: RNase BN is both an exo- and endoribonuclease

Abstract

Processing of the 3' terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3'-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3'-CCA sequence or a 3'-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3' fragments as short as 4 nucleotides. Although the presence of a 3'-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.

FIGURE 1.

Comparison of single- versus double-stranded RNA cleavage by RNase BN. The reactions were carried out as described under “Experimental Procedures” with the addition of 1.5 μg of RNase BN and 10 μm 5′-³²P-labeled single- or completely double-stranded A₁₇ substrate. Portions of 5 μl were taken at 5, 10, and 20 min, as indicated. The value at the bottom of each lane represents the percentage of initial substrate remaining.

FIGURE 2.

Effect of different length of 3′-overhang on RNase BN activity. The reactions were carried out as described for Fig. 1 except that the amount of enzyme was 1.6 μg. Portions of 5 μl were withdrawn after 5, 10, 20, and 30 min as indicated at the bottom of each lane.

FIGURE 3.

RNase BN action on RNA homopolymers. The assay conditions were the same as described in Fig. 1. A, RNase BN was present at 1.5 μg. B, RNase BN was present at 4.5 μg for U₁₇ and 7.5 μg for C_17. The value at the bottom of each lane represents the percentage of initial substrate remaining.

FIGURE 4.

Effect of CCA sequence at the 3′ end of RNA on RNase BN activity. Two different RNA substrates were constructed, one containing the CCA sequence at the 3′ terminus of [³²P]G₅A₁₂ and one containing five A residues following the CCA. Duplex RNAs were annealed with the unlabeled complementary strand, U₁₂C₅, as described under “Experimental Procedures.” The reactions were carried out as described in Fig. 1. A, cleavage of single-stranded G₅A₁₂CCA and single-stranded G₅A₁₂CCA-A₅. B, cleavage of duplex G₅A₁₂CCA and duplex G₅A₁₂CCA-A₅ with 3′-overhang. C, cleavage of duplex G₅A₁₂.

FIGURE 5.

Length requirement for RNase BN digestion. Four substrates of different lengths, A₁₇, A₁₀, A₈, and A₆ were assayed for 30 min as described in the legend to Fig. 1. The value at the bottom of each lane represents the percentage of initial substrate remaining.

FIGURE 6.

Effect of 3′-phosphoryl group on RNase BN activity. Single-stranded (A) and double-stranded (B) A₁₇ and A₁₆P were assayed as described in the legend to Fig. 1. The value at the bottom of each lane represents the percentage of initial substrate remaining.

FIGURE 7.

Mode of action of RNase BN. usRNA1 was assayed as described under “Experimental Procedures.” A, 5′-³²P-labeled usRNA1. B, usRNA1-[³²P]pC. C, usRNA1-[³²P]pCp. D, diagram of endo- and exo-cleavages by RNase BN on usRNA1. Length markers are noted on the right side of each panel.