Previous evidence had suggested the existence of at least two pathways for tRNA maturation in B.subtilis: an RNase Z-dependent endonucleolytic pathway limited to CCA-less tRNA precursors and a second pathway for those containing a CCA motif. In this paper, we show that, while long 3′ extensions of CCA-containing tRNA precursors can be matured by multiple redundant exoribonucleases in vivo, RNase PH plays a major role in degrading the last few nucleotides before the acceptor stem. We show that, similar to RNase Z, which is inhibited by the presence of the CCA motif at the end of the acceptor stem, RNase PH also appears to be sensitive to this sequence and is surprisingly slow to remove the last nucleotide to generate the functional tRNA.
CCA-containing precursors a few nucleotides longer than the mature tRNA accumulated in rph
mutant strains in vivo
, for all of the tRNAs we examined. This is in sharp contrast to E.coli
, where effects of the rph
mutation were only visible on one of the eleven tRNA species examined, or upon precursor tRNA overproduction (2
). The accumulation of precursor species in E.coli
generally required removal of a second exoribonuclease, RNase T. The increased relative importance of RNase PH for removal of the last few nucleotides in B.subtilis in vivo
may be due to the lack of an RNase T ortholog to provide this overlapping function.
The presence of multiple exonuclease mutations in one strain led to increased length of CCA-containing tRNA precursors in vivo
, suggesting that, as in E.coli
, multiple redundant enzymes are involved in the exonucleolytic pathway of tRNA maturation in B.subtilis
. The quadruple deletion strain, rnr yhaM rph pnpA
, grows slowly (doubling time of ~60 min in rich medium). Whether the level of accumulation of tRNA precursors seen in this strain is sufficient to account for its slow growth phenotype remains to be seen. It is clear, however, that a significant level of tRNA maturation still occurs in the quadruple exoribonuclease mutant, suggesting that at least one other enzyme of this pathway remains at large. Preliminary experiments have suggested that recently identified ribonucleases encoded by the B.subtilis ykqC
) or the kapD
gene, encoding a homolog of Homo sapiens
3′ hExo and Caenorhabditis elegans
ERI-1 RNases (29
), are not responsible for this activity (data not shown).
The size of the precursor tRNA that accumulated in vivo in strains lacking RNase PH in was dependent on the tRNA. The trnD-Ser precursor, for example, had only 1–2 extra nucleotides, whereas the trnB-Leu1 precursor was clearly a few nucleotides larger. Since these two tRNA precursors showed no difference in their degradation profiles by purified RNase PH in vitro (), we presume this difference in precursor size observed in vivo is owing to variations in sensitivities of these tRNAs to the remaining processing enzyme(s).
The concentration of RNase PH (38 nM) necessary for the kinetic experiments, even though limiting relative to the substrate (200–300 nM), was quite high, presumably because of the relatively high Km
of RNase PH [1 μM for the E.coli
)] for tRNA. At limiting RNase PH concentrations, the first site of RNase PH arrest detected in vitro
is 3–4 nt from the discriminator base. Curiously, relatively little of the +2 species is seen in the CCA-containing precursors compared to those without CCA (), suggesting that if the CCA sequence is attacked by RNase PH, it has a tendency to remove all but the final cytosine (+1). Should this occur in vivo
, the tRNA could be repaired by nucleotidyl transferase by adding CA to the C at +1. For the CCA-less precursors, significant quantities of the +3, +2 and +1 products are seen, whereas no discriminator product accumulates, i.e. degradation to nt 0. None of these products would be a substrate for nucleotidyl transferase activity, which adds either to the discriminator base or to C- or CC-ends. In this case, RNase Z would be required to cleave endonucleolytically at the discriminator base, generating a viable substrate for nucleotidyl transferase activity.
RNase PH hesitates before removing the +4 nt specifically from CCA-containing tRNA precursors in vitro
. This is in agreement with results in E.coli
that show that, while RNase PH can easily remove the +5 nt from tRNA precursors, it is significantly less efficient at removing the +4 nt than RNase T (32
). The dynamics of removal of the last nucleotides of CCA-less tRNAs has not been addressed with E.coli
RNase PH, mainly because this organism possesses only tRNAs with encoded CCA. Since RNase PH can degrade CCA-less precursors to +3, this would suggest that the phenomenon is sequence-, rather than position-dependent. This is not an artefact of the sequence we chose to replace the CCA motif in the experiment shown in , since RNase PH behaves in the same manner when the CCA motif is replaced with a sequence other than UAA in the case of trnI-Thr
(data not shown). It is not clear why a check-point at +4 for CCA-containing tRNAs would be a beneficial property of RNase PH or, indeed, whether this is a significant event in vivo
where other enzymes are present. The distributive nature of RNase PH degradation close to the acceptor stem, also observed in the E.coli
), would allow other, possibly more efficient enzymes, access to the substrate at each successive round of nucleotide removal. The active site of B.subtilis
RNase PH is deep within the enzyme, at the base of a cleft that can accommodate the CCA motif and the first few base pairs of the acceptor stem (34
). Thus, the potential for sensing the presence of the CCA motif clearly exists.
Although RNase PH is sensitive to the CCA motif in vitro
, this only appears to concern the removal of the very last nucleotide 3′ to the CCA motif; 3′ extensions of CCA-less precursors are also degraded by RNase PH in vitro
( and ). What then accounts for the in vivo
observation that the rph
and other exonuclease mutations only affect the processing of CCA-containing tRNAs? This result could be explained by an inhibition of exonucleases by secondary structures downstream of the majority of CCA-less tRNAs, making these precursors substrates primarily for RNase Z (). Out of the 27 CCA-less tRNA precursors in B.subtilis
, 17 are predicted to have transcription terminators (13 species) or stable stem–loops (4 species) protecting their 3′ ends from exonucleolytic degradation (data not shown). A good example of the inhibitory effect of a terminator structure on RNase PH degradation in vitro
was seen with the trnSL-Ala1
tRNA in . We believe that the 10 remaining CCA-less tRNAs can be processed either endonucleolytically or exonucleolytically and would, thus, not be predicted to accumulate as precursors in the absence of only one pathway. Indeed, three CCA-less tRNAs (trnD-Cys
) were identified by Pellegrini et al
) that were still correctly matured under conditions of RNase Z depletion. In their primary transcripts, these tRNAs are followed closely by other tRNA species and are expected to have short unprotected 3′ extensions upon processing of the 5′ side of the downstream tRNA by RNase P, permitting maturation either by the exonucleases or by RNase Z. Thus, the two pathways of tRNA maturation in B.subtilis
are not likely to be fully mutually exclusive; 17 tRNAs are predicted to be processed primarily by RNase Z, 59 by exonucleases and 10 by either pathway.
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 (more ...)