Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

Abstract

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.

Figure 1

Northern blots of total B.subtilis RNA in various ribonuclease mutants probed for individual tRNAs. Genotypes are listed in Table 1. The tRNAs probed are indicated by their gene names above each blot. tRNA^Gly1 is encoded by four individual genes trnB, trnD, trnI and trnJ. Precursor species are indicated by closed triangles; mature species by open triangles. The presence (+CCA) or absence (−CCA) of an encoded CCA motif is indicated below the blot for each tRNA species.

Figure 2

Northern blots of total B.subtilis RNA in exoribonuclease mutants probed for individual tRNAs. Genotypes are listed in Table 1. The tRNAs probed are indicated by their gene names above each blot. tRNA^Gly1 is encoded by four individual genes trnB, trnD, trnI and trnJ. Precursor species are indicated by closed triangles; mature species by open triangles. The presence (+CCA) or absence (−CCA) of an encoded CCA motif is indicated below the blot for each tRNA species; (+/−CCA) indicates that, for a given tRNA, some genes have an encoded CCA, while others do not.

Figure 3

Degradation kinetics of tRNA 3′ extensions by RNase PH in vitro. The full-length transcript is indicated by FL. The T7 RNA polymerase pause/termination site downstream from the tRNA 3′ end is indicated by +6 or +7. The transcription terminator (ter) of trnSL-Ala1 and stem–loop (SL) of trnD-Cys are also shown. The sizes of the various species are given in parentheses. The full-length transcripts are larger than the natural 3′ ends of the transcripts in vivo and were chosen for their suitability for oligonucleotide hybridization for template amplification by PCR. The position of the discriminator base (arrowhead) was identified for each tRNA species by cleavage of the precursor by 14 ng/μl RNase Z (lane Z) for 15 min as described by Pellegrini et al. (20). For those tRNA precursors containing a CCA motif, short variants were made lacking a CCA motif, permitting cleavage by RNase Z. Incubation times are given in minutes. The zero minute time point is in the absence of RNase PH.

Figure 4

Effect of CCA motif on RNase PH degradation kinetics. (A) tRNA precursor substrates showing positions of nucleotides relative to discriminator base (N₀). All precursor tRNAs were designed to have 6 nt (+6) 3′ extensions. The CCA-less variants have a UAA motif instead of the CCA. (B) Degradation kinetics of trnI-Thr tRNA precursors, with and without a CCA motif, by RNase PH in vitro. Similar reactions are shown for (C) trnD-Ser and (D) trnB-Leu1. The arrowhead indicates the position of the discriminator base of the tRNA, identified by cleaving the −CCA version of the tRNA precursor with RNase Z (lane Z). Incubation times are given in minutes. The zero minute time point is in the absence of RNase PH. The position of the major stalling site is indicated as +3 or +4 to the right of each autoradiogram.

Figure 5

Model for 3′ maturation of tRNAs in B.subtilis. In the maturation of CCA-less tRNAs by the endonucleolytic pathway, exoribonucleases (pac-man symbol; exos) are inhibited (indicated by the X) by 3′ terminal structures and these tRNAs are thus primarily substrates of RNase Z (scissors). The 3′ trailer fragment generated by RNase Z is presumably eventually degraded by exonucleases in a much slower reaction. CCA-less tRNA precursors that are not protected by secondary structures can be degraded by either pathway. In the maturation of CCA-containing tRNAs, RNase Z activity is inhibited by the CCA motif, and the 3′ extension is degraded by exonucleases. Should part of the CCA sequence be removed by the exonuclease, it is repaired by nucleotidyl transferase (CCase).