Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

Abstract

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.

Fig. 1. Sequence comparison of potential B.subtilis, E.coli and M.jannaschii RNase Z orthologues. Identical amino acids are marked with dark shading. Similar amino acids have lighter shading. The phosphodiesterase (PDE) domain (thick overline), proposed zinc finger motif (asterisks) and β-lactamase domain (thin overline) (Schiffer et al., 2002) are indicated.

Fig. 2. Processing of B.subtilis threonine tRNAs under conditions of decreased yqjK expression in vivo. (A) Northern blot of RNA isolated from two independent clones (1 and 2) of SSB320 in the presence (+) and absence (–) of IPTG, probed for trnI-Thr with oligo HP262. (B) Northern blot of RNA isolated from the same clones, probed for trnB/D/J/SL-Thr with oligo HP263. Accumulating precursor species are marked with arrowheads. The positions of a DNA marker are indicated in nucleotides.

Fig. 3. Processing of 11 B.subtilis tRNAs in the absence of yqjK expression in vivo. The autoradiogram shows a northern blot of RNA isolated from SSB320 in the presence (+) and absence (–) of IPTG, probed for the tRNAs indicated. Some probes hybridize to redundant tRNA genes, e.g. trnB/D/I/J-Gly1. Precursor species (arrowheads) accumulate for five tRNA species. Those tRNAs with an encoded CCA motif are indicated under the autoradiogram. The trnD-Asn gene has an encoded CCA motif, while trnI-Asn does not. The positions of a DNA marker are indicated in nucleotides.

Fig. 4. The tRNA precursors that accumulate under conditions of decreased yqjK expression have 3′ extensions. The northern blots for the Val2-trnSL, Ala1-trnSL, Arg-trnQ and Leu2-trnS tRNAs (tRNA) shown in Figure 3 were re-probed with oligos specific for the 3′ extensions (3′). Only the –IPTG samples are shown. The precursor species are indicated by arrowheads.

Fig. 5. (A) Purification of His-tagged YqjK from B.subtilis. Lane 1, extract from uninduced culture; lane 2, extract from culture induced with 0.5 mM IPTG; lane 3, soluble fraction from extract of induced culture; lane 4, purified His-tagged YqjK (RNase Z). A molecular weight marker (in kDa) is shown in the lane marked M. (B) Processing of Oenothera berteriana mitochondrial pre-tRNA^Tyr (Pre) by 2.5 ng/µl Arabidopsis thaliana (At) nuclear RNase Z (nuz) and 3.5 ng/µl B.subtilis YqjK (Bs). A molecular weight DNA marker is shown in the lane marked M. The different precursor molecules and products are symbolized at the sides of the autoradiogram. (C) Labelling of RNase Z-processed tRNA^Tyr with [α-³²P]pCp. tRNA^Tyr, produced by maturation of pTyr by B.subtilis YqjK (Bs) and A.thaliana nuz (At), was incubated with [α-³²P]pCp and RNA ligase.

Fig. 6. Processing of trnI-Thr (A) and trnB-Thr tRNA (B) precursors by RNase Z in vitro. Precursor tRNAs with 83 and 47 nucleotide trailer sequences, respectively, were synthesized by T7 RNA polymerase in vitro (see Materials and methods). The right-angled triangle indicates the direction of increasing enzyme concentration: 0, 0.15, 1.5, 15 and 150 ng/µl RNase Z. An RNA species (asterisk), resulting from premature transcription arrest of T7 RNA polymerase approximately six nucleotides downstream of both tRNA structures, is also a substrate for RNase Z. The different precursor molecules and products are symbolized at the sides of the autoradiograms. The double band corresponding to the trailer species in (A) is likely to be due to the tendency of T7 RNAP to add one or two uncoded nucleotides to the 3′ end of transcripts. (C) Mapping of the RNase Z cleavage site on trnI-Thr pre-tRNA. The autoradiogram shows the reverse transcriptase reaction (RT) using primer HP62 and the corresponding sequencing reaction (lanes A, C, G and T). A portion of the sequence is given to the right of the gel. The two short arrows indicate the sites of RNase Z cleavage. The 3′ end of the mature tRNA sequence is indicated.

Fig. 7. Effect of the CCA motif on processing of trnI-Thr precursor tRNA by RNase Z. Native and mutant precursor tRNAs with six nucleotide 3′ trailer sequences were synthesized by T7 RNA polymerase, taking advantage of the natural transcription termination at this site. RNase Z processing assays of trnI-Thr precursor with native (A) TAAATG and mutant (B) CCAATG, (C) TCAATG, (D) CAAATG trailer sequences downstream of discriminator base. The V_max and K_m values of each of the substrates for RNase Z are as follows: TAAATG (V_max 52.4 fmol/min; K_m 0.97 µM), CCAATG (V_max 0.23 fmol/min; K_m 2.4 µM), TCAATG (V_max 33.8 fmol/min; K_m 0.58 µM), CAAATG (V_max 0.34 fmol/min; K_m 2.2 µM). The direction of increasing enzyme concentration (0, 0.15, 1.5, 15 and 150 ng/µl RNase Z) is given by the right-angled triangles. The precursor molecule and mature tRNA are symbolized at the sides of the autoradiograms. In (A), a band the same size as the cleaved product, visible in the absence of added RNase Z, is due to transcription arrest/pausing at this site by T7 RNA polymerase.

Fig. 8. Effect of 5′ extensions on processing of trnI-Thr precursor tRNA by RNase Z. Precursor tRNAs were synthesized with 83 nucleotide 3′ trailers and 5′ extensions of (A) 66, (B) 33, (C) 10, (D) 5, (E) 1 nucleotide or (F) no extension. The various precursor molecules, mature tRNA and 83 nucleotide 3′ trailer are symbolized to the right of each autoradiogram. The direction of increasing enzyme concentration (0, 0.15, 1.5, 15 and 150 ng/µl RNase Z) is given by the right-angled triangles. In (E) and (F), a band the same size as the cleaved product, visible in the absence of added RNase Z, is due to transcription arrest/pausing at this site by T7 RNA polymerase.