Our initial goal in purifying additional RNases from B. subtilis
was to identify those activities responsible for initiation and completion of bulk mRNA decay. Earlier studies with B. subtilis
strains with deletions of the genes encoding PNPase and RNase R, the two 3′-to-5′ exoribonucleases identified previously, indicated that these RNases are dispensable with regard to bulk mRNA decay (48
). YhaM, the RNase isolated in the present study, turned out to be an interesting exoribonuclease activity that is unique to gram-positive organisms. Our findings confirm the recent speculation of Aravind and Koonin (5
), based on predictions of protein domain structure, that YhaM is an RNase. However, because the in vivo function of YhaM remains to be determined, we prefer to defer renaming the yhaM
gene and the protein it encodes.
We observed no growth phenotype or significant change in bulk mRNA half-life associated with the absence of YhaM. The absence of YhaM in a strain that also lacks PNPase and RNase R did result in a failure to grow at room temperature and an extended lag phase at 37°C. The absence of individual RNases also affected growth at a low temperature (Table ), but not as severely as the triple RNase mutant. It has been reported that the cold shock protein cspA
of E. coli
, which was named because of its massive induction of expression upon cold shock (28
), is also induced when a stationary-phase culture is diluted with fresh medium (12
). Thus, the phenotypes of the triple mutant that we observed (cold sensitivity and extended lag phase) may be related.
In E. coli
, expression of PNPase is induced by cold shock (31
), and a pnp
strain of E. coli
is severely deficient in colony formation at temperatures below ambient temperature (35
). It has been shown recently that the function of PNPase in growth at cold temperatures is to participate in turnover of mRNAs that are involved in the cold adaptation program (50
). Since B. subtilis
homologues of the major E. coli
cold shock proteins are also regulated posttranscriptionally (32
), the increased cold sensitivity of the B. subtilis
triple RNase mutant may suggest that YhaM is indeed involved in at least some aspects of mRNA decay.
After preparation of this article, a report by Noirot-Gros et al. on protein interactions of the B. subtilis
DNA replication apparatus was published (39
). Using yeast two-hybrid screening, these authors found a specific interaction of YhaM with DnaC, a DNA helicase that is a component of the replisome. It is possible that the single-stranded DNase activity of YhaM, which we have shown here, could be involved in DNA replication. On the other hand, based on the similarities of YhaM and the S. aureus
CBF1 protein, we can speculate that the relevance of YhaM to DNA replication may be a double-stranded DNA binding function. Although we showed that CBF1 protein has exoribonuclease activity that is similar to that of YhaM in its divalent cation dependence, CBF1 was first described as a DNA-binding protein specific for the cmp
region of plasmid pT181 (52
). While it might seem surprising that an exoribonuclease would have double-stranded DNA-binding capacity, there is a precedent for this observation. It has been shown that E. coli
PNPase also can bind specific double-stranded DNA sequences (51
The properties of purified YhaM are of interest. First, we can rule out the possibility that YhaM is one of the previously isolated hydrolytic B. subtilis
RNases. The descriptions of two intracellular RNases that have been reported clearly differentiate those activities from YhaM: Kennell and colleagues reported a pyrimidine-specific endoribonuclease that had a molecular size of about 15 kDa, was active in the presence of Mg2+
, and was unable to degrade single-stranded DNA (36
). An earlier report described an intracellular exoribonuclease that was present at low levels in vegetative cells and was induced in sporulating cells (33
). The size of this protein was estimated to be 72 kDa.
Second, while an absolute requirement for divalent cations (usually Mg2+
) is common among the RNases (53
), the dependence on Mn2+
) is quite unusual. Deutscher and colleagues have reported that E. coli
RNase BN, a 3′ exoribonuclease specific for tRNA, is most active in the presence of Co2+
). However, both Mg2+
could also serve as cofactors for RNase BN. We could not detect any YhaM RNase activity in the presence of a wide range of Mg2+
concentrations. YhaM was active in vitro at low levels of Mn2+
(10 μM), which may be within the range of intracellular Mn2+
concentrations (the actual free Mn2+
concentration is not known).
Third, this is the first report of an RNase that combines two domains—the OB-fold and the HD domain—that are otherwise independently associated with proteins engaged in ribonucleotide biochemistry. None of the proteins annotated as “RNase” in the SWISS-PROT and TrEMBL databases contains an OB-fold that is similar to the one in YhaM. While the S1 domain, which is present in several RNases, is a member of the OB-fold superfamily (14
), the YhaM 17-90 region does not resemble an S1 domain; furthermore, the OB-fold in the S1 domain does not resemble the one in YhaM. In addition, none of the S1-containing proteins has an HD domain. Although RNase T of E. coli
is similar to YhaM in that it also is able to degrade single-stranded DNA exonucleolytically (53
), we have searched for but have not found evidence that YhaM is related to RNase T at the structural or sequence level. Thus, the predicted domain structure of YhaM and the other CBF1 proteins is unique.
The predicted structure of the YhaM OB-fold very clearly suggests this as a single-stranded nucleic acid binding protein, with the exposed aromatic side chains likely to be critical for binding (37
). It would be interesting to test whether the W51 and Y76 residues are important for the ability of YhaM to recognize both RNA and single-stranded DNA as substrates. Based on the sequence similarity and modeling calculations, YhaM and its homologues appear to have one OB-fold domain in the 17-90 region and one HD domain in the 160-279 region (YhaM numbering), as shown in Fig. . The region between the OB-fold and HD domains does not match any region of known structure or function in proteins outside this group of OB-plus-HD proteins. The region from position 147 to position 160 contains several highly conserved positions. This may be an extension of the HD domain (light green block in Fig. ), which would make the interdomain region even shorter. In addition, the OB-fold domain may cover more than the 17-90 region, because the remote sequence similarity match used to define it is likely to correspond only to the most conserved core of the domain, leaving more-variable regions undetected. With the present data we cannot distinguish absolutely whether the 91-146 interdomain region is merely a linker connecting the OB-fold and HD domains or is a third domain in the OB-plus-HD family, but the small size of this region and the lack of a match to any domains of known structure or function make the latter possibility less likely.
From the bacterial genomes already sequenced, we found that YhaM/CBF1 binding protein sequences are present only in gram-positive organisms. As mentioned above, the B. subtilis
genome lacks clear sequence homologues with several important E. coli
RNases—RNase E, RNase II, and oligoribonuclease. In addition, it has been shown that RNase III is essential in B. subtilis
), although it is dispensable in E. coli
). Thus, the discovery of YhaM further highlights potential differences in the nature of nuclease activities found in gram-negative and gram-positive organisms.