OLE RNA, an RNA motif that is highly conserved in several extremophilic bacteria, is a substrate for and can be regulated by RNase P RNA
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Contributed by Sidney Altman, March 27, 2007 (received for review January 31, 2007)
Abstract
OLE (ornate, large, and extremophilic) RNA is a noncoding RNA that is found in several extremophilic bacteria, including Bacillus halodurans. The function of OLE RNA has not been clarified. In this study, we found that RNase P cleaves OLE RNA and that the cleavage leads to a small reduction of expression of a downstream gene determined by analyses in vitro and in vivo. Under RNase P-deficient conditions, the amount of OLE RNA increased. Our results imply that RNase P could play a role in the regulation of gene expression in relation to conserved RNA motifs like OLE RNA as well as in riboswitches and operons.
OLE (ornate, large, and extremophilic) RNA is a highly conserved RNA motif that is found in several extremophilic Gram-positive eubacteria (1). It was identified with bioinformatic approaches for finding additional riboswitches. However, OLE RNA has different biological characteristics from those of riboswitches and is believed to be a different type of RNA element that is involved in gene regulation (1). In Bacillus halodurans, an extremophilic bacterium widely used for industrial purposes, OLE RNA is located between the BH2780 and BH2781 genes (Fig. 1). Although the functions of OLE and BH2780 RNAs have yet to be understood, OLE RNA can form a complex in vitro with the BH2780 protein, which is a possible transmembrane protein, suggesting that OLE RNA might play a role in a fundamental cellular process as a ribonucleoprotein complex (R. Breaker, personal communication). It is also unknown whether OLE RNA regulates the expression of downstream genes, and it is not apparent that the transcript of OLE RNA extends through the beginning of the BH2780 gene.
Gene location of OLE in B. halodurans. (A) Schematic representation of OLE. (B) Nucleotide sequence of OLE and BH2780. The 58th nucleotide of the intergenic region between BH2781 and BH2780 is numbered +1. The position of +678 corresponds to the 735th nucleotide of the intergenic region. The putative −35 and −10 regions are underlined. The OLE sequence is in bold, and the sequence of BH2780 is in italics.
RNase P is a well known endoribonuclease that is responsible for the 5′ maturation of tRNA and several RNA molecules, including 4.5S RNA and operons in eubacteria (2). Besides these substrates, RNase P cleaves transient structures of some riboswitches, such as coenzyme B12 riboswitch in Escherichia coli (3, 4). Here, we show that RNase P cleaves OLE RNA and that this cleavage can affect the expression of a downstream gene in vivo. This kind of RNase P function might be more general than is anticipated in bacterial species.
Results
Riboswitches are RNA elements regulating gene expression downstream of these elements by responding to metabolites, which are produced by the downstream gene product (4). In a previous report, we showed that cleavages by RNase P lead to the reduction of downstream genes in an operon by using lacZ as a reporter gene (3).
β-Galactosidase Activity in Wild-Type and Mutant Strains of RNase P in E. coli.
The effects of the cleavages by RNase P on OLE RNA in vivo were investigated by using a model system with a lacZ reporter gene (Fig. 2). The plasmids pOLE-lacZ and pBAD-lacZ, a control plasmid, were introduced into NHY312 (wild-type) and NHY322 (RNase Pts) strains. A wild-type strain, MG1693, and the mutant strain of RNase E, SK5665, also were transformed to identify the effects of one of the major endoribonucleases in E. coli (5). The expression of OLE RNA was induced by adding 0.2% arabinose to the media, and β-galactosidase activities were measured in the strains at both 30°C and 43°C (Table 1). NHY322 and SK5665 are temperature-sensitive strains; therefore, at 43°C, the relevant enzymes lose their activities.
Construction of the plasmid for the analysis of β-galactosidase activity. The OLE RNA from B. halodurans and lacZ were inserted into the region between the EcoRI and XbaI sites of pBAD18. The lacZ contains the Shine–Dalgarno sequence required for translation. pBAD18 has an arabinose-inducible promoter upstream of the cloning sites. S/D represents the Shine–Dalgarno sequence. pBAD-lacZ also is shown in schematic form.
β-galactosidase activities in E. coli with the OLE RNA
The β-galactosidase activity of NHY322 at 43°C increased 1.6-fold compared with that at 30°C, whereas the activity of NHY312, the wild-type strain, increased 1-fold. The standard deviation of the ratios at different temperatures indicates that these values are distinctly separate, although the magnitude of their difference is not very large. In the case of RNase E, the effects were much greater than those of RNase P. In the SK5665 strain, the β-galactosidase activity increased 2.3-fold at 43°C compared with that at 30°C, whereas the activity increased only 1.1-fold in the wild-type strain, MG1693 (Table 1). From these results, we found that RNase P cleavage on OLE RNA leads to reduced lacZ expression. This phenomenon might regulate gene expression downstream of OLE RNA in other bacterial species as well as in E. coli.
Cleavage of OLE RNA by E. coli RNase P.
OLE RNA was prepared by transcription in vitro. The sequence of the transcript contained the 58th to 735th nucleotides of the intergenic region between BH2780 and BH2781 (Fig. 1 B). By comparing the cleavage pattern of internally labeled OLE RNA with that of 5′ end-labeled OLE RNA, we found that there was one cleavage site in OLE RNA when M1 RNA was used as the cleavage enzyme and two major cleavage sites existed when E. coli RNase P holoenzyme was used (Fig. 3 A). To determine the locale of the cleavage sites by RNase P, we performed primer extension experiments (Fig. 3 B and C) using two individual oligonucleotides that are complementary to different regions. The results showed that M1 RNA cleaves the site between C251 and C252 and that RNase P holoenzyme cleaves the site between A403 and U404 as well as the same site that M1 RNA cleaves. However, experiments with two-dimensional thin-layer chromatography indicate that there is no 5′ phosphate on the 3′ cleavage product at the site between A403 and U404 (data not shown). Either a contaminating phosphatase was present or another ribonuclease was involved with this site.
Activity of E. coli RNase P and M1 RNA on OLE RNA. (A) Cleavage by M1 RNA or RNase P holoenzyme from E. coli. Reactions were conducted as described in Materials and Methods with 5,000 cpm of labeled substrates. The lanes of con, M1, and P represent untreated RNA sample, reaction product with M1 RNA, and reaction product with RNase P holoenzyme, respectively. α-OLE, 5′-OLE, and pSupS1 represent internally labeled OLE RNA, OLE RNA labeled at 5′ end, and internally labeled pSupS1 RNA, respectively. (B) Determination of cleavage sites by primer extension with OLEP1 primer. The lanes of con, M1, and P indicate the extension reactions from untreated OLE RNA alone, reaction product with M1 RNA, and reaction product with RNase P holoenzyme, respectively. The cleavage site is indicated with an arrow. (C) Mapping of cleavage site with primer OLEP2.
Cleavage of OLE RNA by Bacillus subtilis RNase P.
Because the OLE RNA was detected in B. halodurans as well as in several extremophiles, the effects of RNase P on OLE RNA should be determined in an extremophile. However, introducing foreign DNA into B. halodurans is difficult. This problem may arise from the absence of necessary genes for natural competence in B. halodurans in comparison with B. subtilis (6). Therefore, the issue initially was circumvented by substituting B. subtilis (not an extremophile) for B. halodurans. RNase P was reconstituted by mixing RNase P RNA and the cognate His-tagged RNase P protein from B. subtilis. The B. subtilis RNase P cleaved OLE RNA at one site (Fig. 4 A), which was the same as in E. coli, between C251 and C252 (Fig. 4 B). When RNase P RNA alone was used for the reaction, the cleavage pattern was the same as that of the RNase P holoenzyme. The cleavage at the site between A403 and U404, which was seen in E. coli, could not be observed with B. subtilis RNase P.
Activity of B. subtilis and B. halodurans RNase P on OLE RNA. (A) RNase P reaction with E. coli and B. subtilis RNase P. Reactions were performed with 10,000 cpm of labeled OLE RNA or pSupS1 as substrates. In lane P, 50 nM M1 RNA and 500 nM C5 protein were used. In lane BS, 50 nM B. subtilis RNase P RNA and 500 nM B. subtilis RNase P protein were used, whereas in lane BR, 500 nM B. subtilis RNase P RNA was used for the reaction. α-OLE, 5′-OLE, and pSupS1 represent internally labeled OLE RNA, OLE RNA labeled at 5′ end, and internally labeled pSupS1 RNA, respectively. (B) The cleavage sites of B. subtilis RNase P on OLE RNA. Primer extension reaction was conducted as described in Materials and Methods with the OLEP1 primer. The lanes of con, P, BS, S30, and BH indicate the extension reactions from untreated OLE RNA alone, reaction product with E. coli RNase P, reaction product with B. subtilis RNase P, reaction product with B. halodurans S30 fraction, and reaction product with B. halodurans RNase P holoenzyme, respectively. The cleavage site is indicated with an arrow. (C) RNase P reaction with B. halodurans S30 fraction and RNase P. Reactions were performed by using 10,000 cpm of labeled OLE RNA or pSupS1 as substrates. In lane BS, 50 nM B. subtilis RNase P RNA and 500 nM B. subtilis RNase P protein were used, whereas in lanes S30 and BH, 1 μl of fraction was used for the reaction. (D) The cleavage site of B. halodurans RNase P. The cleavage site was determined by primer extension reaction as described for B, except that OLEP3 primer was used. The reaction products were separated on a 6% polyacrylamide gel.
Cleavage of OLE RNA by Partially Purified RNase P of B. halodurans.
An S30 fraction of B. halodurans was fractionated on a DEAE-Sepharose column to purify RNase P through its first convenient step. The active fractions were used for analysis of OLE RNA [see supporting information (SI) Fig. 7]. The main cleavage site was between U462 and U463, although the cleavage band is broad and may indicate two cleavage sites, one also between A461 and U462 (Fig. 4 C and D), which are different from those sites of E. coli and B. subtilis RNase P. B. halodurans RNase P may have a different specificity for OLE RNA.
To verify that the BH cleavage(s) is caused by RNase P, the 5′ terminus of the 3′ cleavage product was analyzed by two-dimensional thin-layer chromatography. If the substrate was labeled either with [α-32P]UTP or with [α-32P]ATP, the 5′ phosphate of the 3′ cleavage product was determined to be pUp (data not shown). RNase P produces 5′ phosphates at its cleavage site, which is a characteristic of its reaction. This observation is strong evidence that RNase P is involved in the BH cleavage of OLE RNA. Cleavage of a transient structure of OLE RNA is discussed herein.
The possibility cannot be excluded that the enzyme fraction that we used here may not have been sufficiently pure, although we observed only one primary cleavage site. One other possibility is that RNase E is involved in this cleavage, because it also produces 5′ phosphates in its 3′ cleavage product. This prospect is unlikely because RNase E elutes at a lower ammonium chloride concentration on DEAE-Sepharose than does RNase P (SI Fig. 7) (7).
Expression of BH2780 in the RNase P-Deficient Strain of B. subtilis.
Because RNase P cleavage can affect the expression of a gene downstream of OLE RNA in E. coli (Table 1), the question remains as to whether RNase P affects the natural downstream gene, BH2780, in B. halodurans. OLE RNA and BH2780 sequences were introduced into the amyE locus of the B. subtilis, our surrogate for B. halodurans, by double crossover recombination (8). The existence of the sequence was confirmed by PCR amplification of genomic DNA (see SI Fig. 8). Because the SSB–OLE–BH strain (in Materials and Methods) has the isopropyl β-d-thiogalactoside (IPTG)-inducible promoter for RNase P RNA, an RNase P-deficient condition could be made by changing the medium to one lacking IPTG and incubating cells in that latter medium for an additional 2 h.
Total RNAs were prepared from cells that grew in media containing IPTG or lacking IPTG. Northern blotting was conducted to observe the expression pattern with primers complementary to the BH2780 or OLE RNA (Fig. 5). The level of BH2780 mRNA hardly changed under the RNase P-deficient condition compared with that under normal conditions. However, it is surprising that the amount of OLE RNA was greatly increased in the case of RNase P inactivation. The steady-state amount of OLE RNA on the RNase P-deficient condition increased approximately 8-fold compared with that under normal conditions. A band containing both the OLE sequence and BH2780 was not evident in the Northern blot, implying that BH2780 might have a transcription start site of its own and might not be under transcriptional control of OLE RNA.
The effect of RNase P on the expression of OLE RNA and BH2780 in the SSB–OLE–BH strain. Total RNA (4 μg) from cells grown in the media containing IPTG (in the + lane) or lacking IPTG (in the − lane) was separated on a 2% agarose gel. The RNAs were transferred to a nylon membrane and probed with the appropriate primers. The OLE RNA (0.5 fmol) from in vitro transcription was used as a marker. The arrow indicates the position of in vivo OLE RNA.
Discussion
Although small RNAs such as microRNAs have been studied intensively recently, large noncoding RNAs also are involved in a variety of cellular functions, including transcriptional regulation, and are less well understood (9). These RNAs usually bind to their target directly or they require a protein partner for regulating their targets (9). OLE RNA was discovered as a candidate for a riboswitch, but it has quite different biological characteristics from those of riboswitches. The size of OLE RNA is much bigger than those of riboswitches, and the metabolite responding to OLE RNA has not been identified. Therefore, OLE RNA was suggested as a novel gene regulation element (1). In relation to this, the gene product of which is located downstream of OLE RNA, BH2780 in B. halodurans, is known to make a complex with OLE RNA in vitro, and the complex is found in a cellular membrane fraction (R. Breaker, personal communication).
Effects of RNase P on the Expression of Genes Downstream of Cleavage Sites.
In this study, we found that RNase P cleavage of OLE RNA leads to the decrease of expression of the LacZ gene downstream of OLE RNA in E. coli. This effect is similar, but not as great in absolute numbers, to the polarity effect that can be observed in lacYA operon in E. coli. The change of gene expression upstream of OLE RNA was not checked. Besides OLE RNA, it has been reported that RNase P cleavage on Btu, which is a riboswitch in E. coli, leads to down-regulation of a gene downstream of that element (3). Our results increase the possibility that this ancient enzyme, RNase P, may play diverse roles with the RNA elements of gene regulation, including riboswitches and novel elements such as OLE RNA in other bacterial species.
Biogenesis of OLE RNA and the Regulation of BH2780 Expression.
The sequence analysis of the region containing OLE RNA and BH2780 revealed a strong candidate for a transcriptional promoter (Fig. 1 B). The sequence of the −10 region matches the consensus sequence, TATAAT, perfectly and the −35 sequence also has a relatively high homology (10). Considering that a substantial amount of β-galactosidase activity from the cells that contained the pOLE-lacZ plasmid was detected even without the addition of any arabinose, some of the OLE RNA might be made by initiation of transcription from this position. However, according to a previous paper (1), large transcripts containing OLE RNA exist in B. halodurans, which implies that OLE RNA is expressed as part of a large polycistronic mRNA. It is possible to assume that OLE RNA is made as a part of a large transcript, as well as an independent RNA molecule.
The assay in vitro using the S30 fraction of B. halodurans and partially purified RNase P fraction showed there were cleavage sites at the region closer to 3′ end of OLE RNA (Fig. 4 C). This finding implies that some of OLE RNA is made by cleavage of the primary transcripts in cells, which is supported by the fact that the RNA bands in B. subtilis appeared smaller than transcript in vitro of OLE RNA in Northern blot analysis (Fig. 5).
If BH2780 does not have an independent promoter of its own, the transcription of BH2780 mRNA depends exclusively on the transcription events that happen upstream of this gene. If the transcripts do not extend to the region of BH2780, the gene cannot be expressed. In this case, the cleavage at sites upstream of BH2780 might affect the stability of the remaining BH2780 mRNA. Considering our data on the β-galactosidase activity in E. coli, RNase P cleavage may lead to the destabilization of the mRNA downstream because lacZ message has a Shine–Dalgarno sequence within the sequence; therefore, the translation of lacZ mRNA could be possible even after RNase P cleaved the RNA. However, the Northern blot analysis implies that, in B. halodurans, this kind of regulation might not happen. Under the RNase P-deficient condition, the level of BH2780 seems not to be changed compared with that under normal conditions (Fig. 5). Further studies on gene regulation in B. halodurans and related bacteria might explain the events in vivo.
Characteristics of RNase P Cleavage on the OLE RNA.
The cleavage sites are summarized on the putative secondary structure of OLE RNA (Fig. 6 A). The cleavage at the site between C251 and C252 was made by RNase P from both E. coli and B. subtilis, whereas the cleavage at the site between A403 and U404 happened only when RNase P from E. coli was used, although there is some question as to whether or not this is a valid RNase P site. The cleavage site between C251 and C252 is a classical RNase P cleavage site. The cleavage site is on the single-stranded region followed by a rigid stem structure (Fig. 6). However, the cleavage site between A403 and U404 is on the single-stranded region, which looks like a bulge, followed by an internal loop. The cleavage site between U462 and U463 (and A461 and U462) of B. halodurans RNase P follows a stem that is adjacent to the single-stranded region (see below). It is worth mentioning that the RNase P RNA of E. coli, M1 RNA, cleaves OLE RNA only at the site between C251 and C252, which implies that RNase P protein, C5 protein, might confer the ability of cleaving another position of OLE RNA (Fig. 3). We also exchanged RNase P protein of E. coli and B. subtilis and performed the cleavage assay in vitro (see SI Fig. 9). The cleavage pattern by the complex of B. subtilis RNase P RNA and C5 protein looked similar to that of the complex of M1 RNA and C5 protein, whereas the complex of M1 RNA and B. subtilis RNase P protein produced a pattern similar to that of B. subtilis RNase P holoenzyme. For tRNA precursors, it has been reported that the exchange of RNase P protein does not change cleavage patterns (11, 12). RNase P proteins, however, seem to change the specificity of cleavage on OLE RNA by different hybrid RNase Ps.
The cleavage sites of OLE RNA by RNase P. (A) The cleavage sites of RNase P are superimposed on the putative secondary structure, which were provided by R. Breaker. The susceptible nucleotides in the in-line probing experiment are marked in circles. The cleavage sites of RNase P are indicated by arrows. E, BS, and BH represent E. coli RNase P, B. subtilis RNase P, and B. halodurans RNase P, respectively. The unmarked arrow indicates the second E. coli RNase P holoenzyme cleavage site as described in the text. (B) A transient structure with the cleavage by B. halodurans indicated by an arrow. The dots represent cleavage of the intact OLE RNA by RNase T1.
Cleavages of transient structures in riboswitches have been reported recently (3). A similar transient structure of OLE RNA is shown in Fig. 6 B as a substrate for cleavage by partially purified B. halodurans RNase P. A canonical RNase P cleavage is shown in the model.
We did not measure the kinetics of RNase P cleavage on OLE RNA in vitro. Such possible data would have to be complemented by kinetics of RNase E cleavage to get an approximate value of these enzymes in OLE RNA survival in vivo. The data on strains that lack RNase P or RNase E clearly show that OLE RNA is a substrate for both enzymes in vivo.
Function of OLE RNA.
RNase P cleaves a stable noncoding RNA, 4.5S RNA in E. coli (13), which is a component of the signal response particle. It is interesting that RNase P cleaves the RNAs that are located in a membrane fraction and a component of a ribonucleoprotein complex in both E. coli and B. halodurans.
RNase P cleaves the 5′-UTR region of secG, a stimulator of protein translocation, the rbs operon containing rbsDACB and rbsK, and tnaB, which is a low-affinity tryptophan permease (14). In these cases, RNase P cleavage should lead to the low expression of the downstream genes. Whether RNase P plays a specific role in the metabolism relating to membranes in B. halodurans and other extremophilic bacteria is unknown.
The statement that OLE can be regulated by RNase P applies, of course, to E. coli, although the possibility that this can happen in B. halodurans is not out of the question. The experiment with OLE in front of a reporter gene would have to be verified in the extremophile.
Materials and Methods
Strains.
NHY322 [Δ(proB lac) ara, gyrA, thi, zic-501::Tn10, rnpAts] is an isogenic mutant strain that carries the A49 mutation of C5 protein (protein subunit of RNase P) in E. coli (15). NHY312 [Δ(proB lac), ara, gyrA, thi, zic-501::Tn10, rnpA +] is the wild-type parent. For RNase E deficiency, SK5665, a temperature-sensitive strain in RNase E deficiency, was used in some experiments. MG1693 is the wild-type strain for RNase E (16). MG1693 and SK5665 were provided by S. Kushner (University of Georgia, Athens, GA). B. subtilis SSB318 was used as a conditional RNase P-deficient strain (provided by R. Hartmann, Philipps-Universität Marburg, Marburg, Germany) (17). In SSB318, the original promoter of RNase P RNA was substituted by Pspac, an IPTG-inducible promoter. Without IPTG, the transcription from this promoter is substantially reduced. B. halodurans (no. 21591; American Type Culture Collection, Manassas, VA) was used in several experiments.
For the analysis of the RNase P effect on OLE RNA in B. subtilis, the region containing both the OLE sequence and the BH2780 sequence was amplified with PCR by using genomic DNA of B. halodurans and primers of OLE-BH-5 and OLE-BH-3. All primers used in this study are shown in SI Table 2. We confirmed the sequence of the PCR product after every step of PCR amplification. The amplified product was inserted into the sites between EcoRI and BamHI of an integration vector, pDG1661 (8). The resultant vector was named p1661-OLE-BH. Transformation of B. subtilis was performed according to a previous paper (18). Transformed cells were confirmed on LB plates containing 0.5 μg/ml erythromycin, 12.5 μg/ml lincomycin, 5 μg/ml chloramphenicol, and 1 mM IPTG for the SSB318 strain. The resultant strain was named SSB-OLE-BH.
Preparation of OLE RNA in Vitro.
The plasmid that contains T7 promoter and the OLE RNA sequence of B. halodurans was provided by R. Breaker (Yale University). The region from +1 to +678 of OLE RNA as well as T7 promoter was inserted into the EcoRI site of pUC19, creating pUC19-OLE. The 58th nucleotide of the intergenic region between BH2781 and BH2780 is numbered +1. EcoRI-treated pUC19-OLE was used as a template for transcription in vitro of OLE RNA, with T7 RNA polymerase and the template. The resultant RNA is 692 nt long, including 2 additional G nucleotides at the 5′ end and 12 nucleotides originating from pUC19 as well as the OLE RNA sequence.
RNase P Activity Assays.
OLE RNA was internally labeled with [α-32P]GTP during transcription reaction by using EcoRI-treated pUC19-OLE and T7 RNA polymerase. OLE RNA was also labeled at either 5′ end with [γ-32P]ATP and T4 polynucleotide kinase or the 3′ end with [32P]pCp and T4 RNA ligase. For a control reaction, the yeast suppressor precursor tRNASer (SupS1) of yeast was internally labeled with [α-32P]GTP and used as a substrate for RNase P.
Reactions were performed at 37°C in 10 μl of 1× PA (50 mM Tris·HCl, pH 8.0/100 mM NH4Cl/10 mM MgCl2) for E. coli RNase P. When M1 RNA alone was used for the reaction, MgCl2 was added to a concentration of 100 mM. When B. subtilis RNase P was used, MgCl2 was added to a concentration of 60 mM. When B. subtilis RNase P RNA alone was used for the reaction, we used a modified buffer (50 mM Tris·HCl, pH 8.0/600 mM NH4Cl/200 mM MgCl2). Reactions were terminated by adding 10 μl of a 10 M urea/10% phenol solution.
Primer Extension.
The primer extension reaction was performed according to a previous report (19). A total of 0.1 pmol of OLE RNA was incubated with RNase P at 37°C for 30 min. After the reaction time, the mixture was extracted with TE-saturated phenol/chloroform/isoamyl alcohol (25:24:1) and then chloroform/isoamyl alcohol (24:1). The products were recovered by ethanol precipitation and redissolved in 10 μl of water. One-fifth of the products was mixed with 0.2 pmol of the appropriate primer, which was labeled at the 5′ end with [γ-32P]ATP, and incubated at 65°C for 10 min and slowly cooled down to room temperature. SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) was added to the mixture and the extension reaction was performed at 42°C for 1 h. The reaction product was recovered by ethanol precipitation and separated on an 8% or 6% polyacrylamide gel that contained 7 M urea.
Construction of Plasmids for β-Galactosidase Assay.
For the β-galactosidase assay, we constructed plasmids according to a previous report with some modification (19). pBAD18 was used as a template plasmid, and pLZBH was used as a lacZ source (20). We amplified the DNA containing OLE RNA sequence with the primers of OLE-lac-5 and OLE-lac-3. The amplified product was treated with EcoRI and HindIII, and then it was mixed with the lacZ-containing fragment of pLZBH, which was cut with HindIII and XbaI. The mixture was inserted into the sites between EcoRI and XbaI of pBAD18, making the resultant plasmid, pOLE-lacZ (Fig. 2). As a control, the plasmid lacking the OLE sequence was constructed with a similar method. The fragment of pLZBH cut with HindIII was treated with Klenow fragment to make both ends blunt. The fragment was cut with XbaI, and the reaction product was cloned into SmaI–XbaI sites of pBAD18, creating the resultant plasmid, pBAD-lacZ.
β-Galactosidase Assay.
The β-galactosidase assay was performed according to published reports (19, 21). Arabinose (0.2%) was used to induce the transcription from pOLE-lacZ and pBAD-lacZ. Cells containing the appropriate plasmid were cultured overnight at 30°C and diluted 1:100 into fresh media and grown to OD600 = 0.3 at 30°C. The temperature was shifted to 43°C in case of NHY322 or SK5665 and cells were further incubated for 10 min. Arabinose (0.2%) was added to the cultures and, after 60 min, the β-galactosidase assay was performed on cell extracts.
Preparation of RNase P from B. halodurans.
B. halodurans was cultured overnight in LB media containing 1% Na2CO3. Cells were collected by centrifugation at 1,900 × g at 4°C. The pellet was frozen at −80°C and mixed with twice its own weight of alumina. The mixture was ground with a prechilled grinder in a mortar. 1× PA buffer containing 0.1 mM DTT and Complete (one tablet per 50 ml), a protease inhibitor mixture (Roche Applied Science, Indianapolis, IN), was added to the paste as well as DNase (Worthington, Lakewood, NJ) and incubated at 4°C for 30 min. The sample was centrifuged at 7,700 × g for 10 min and supernatant was further centrifuged at 30,000 × g for 40 min to prepare the S30 fraction. The S30 fraction was loaded onto a DEAE-Sepharose column that was preequilibrated with 1× PA buffer. 10 volumes of 1× PA buffer were used for washing the column and then 0.2, 0.3, 0.5, and 0.7 M NH4Cl in PA buffer were applied to the column to elute proteins. The fraction having RNase P activity was concentrated with an Amicon Ultra-15 centrifugal filter with a molecular weight cutoff of 10,000 (Millipore, Billerica, MA).
Northern Blot Analysis.
SSB-OLE-BH was incubated overnight in the LB media containing 0.5 μg/ml erythromycin, 12.5 μg/ml lincomycin, 5 μg/ml chloramphenicol, and 1 mM IPTG. The overnight culture was diluted into the same media with a starting OD600 = 0.05. When OD600 reached 0.3, cells were collected and washed twice with media lacking IPTG and further incubated for 2 h in the media that contained or lacked IPTG. Total RNA was prepared as described previously with some modification. Lysozyme (10 μg/ml) was added to cells, and the cells were kept on ice for 10 min before phenol extraction (22). The RNAs (4 μg) were separated on a 2% agarose gel. Northern blot analysis was performed as described previously (23). Oligonucleotides of +1139R, OLEP1, and 23SR were labeled at their 5′ ends by using [γ-32P]ATP and were then used as probes for BH2780, OLE RNA, and 23S rRNA, respectively.
Acknowledgments
We thank Drs. Roland Hartmann and Sidney Kushner for gifts of relevant strains; Ms. Donna Wesolowski for assistance in the preparation of RNase P from Bacillus strains; and Drs. Ronald Breaker, Elena Puerta-Fernandez, and were colleagues in the S.A. laboratory for helpful discussions. S.A. was supported financially by Yale University.
Footnotes
- *To whom correspondence may be addressed. E-mail: sidney.altman{at}yale.edu
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Author contributions: J.-h.K. and S.A. designed research; J.-h.K. performed research; J.-h.K. and S.A. analyzed data; and J.-h.K. and S.A. wrote the paper.
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The authors declare no conflict of interest.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0701715104/DC1.
- Abbreviations:
- IPTG,
- isopropyl β-d-thiogalactoside;
- OLE,
- ornate, large, and extremophilic.
- © 2007 by The National Academy of Sciences of the USA
