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Originally published In Press as doi:10.1074/jbc.M702747200 on May 16, 2007

J. Biol. Chem., Vol. 282, Issue 28, 20238-20244, July 13, 2007
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Initiation of decay of Bacillus subtilis trp leader RNA*

Gintaras Deikus and David H. Bechhofer1

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine of New York University, New York, New York 10029

Received for publication, March 30, 2007 , and in revised form, May 15, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3' end of trp leader RNA and PNPase digestion of trp leader RNA from the 3' end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified four 3'-to-5' exoribonucleases of Bacillus subtilis that can be involved in the decay of mRNA (1). One of these, PNPase,2 a processive enzyme that degrades RNA phosphorolytically, was shown earlier to be the major exonucleolytic activity in B. subtilis extracts (2), and we have shown more recently that PNPase is the main enzyme responsible for mRNA turnover in vivo (1, 3). A strain with a disruption of the pnpA gene, encoding PNPase, is viable but has a number of phenotypic defects including an inability to regulate transcription of the trp operon (4). In a wild-type strain, the B. subtilis trp operon is regulated at the level of transcription termination (5, 6). When the supply of intracellular tryptophan is low, the trp operon genes are transcribed from a constitutive promoter, and more tryptophan is made. In the presence of ample tryptophan, transcription from this promoter terminates after transcribing the 139-nucleotide (nt) trp leader RNA. Termination is mediated by the trp RNA-binding attenuation protein (TRAP) complex. TRAP is activated by tryptophan itself and binds with high affinity as an 11-mer to 11 trinucleotide repeats located between nt 36 and 91 of the trp leader sequence (Fig. 1B). Binding of the TRAP complex allows a stem-loop transcription terminator structure to form, which consists of nt 108–133 of the trp leader sequence. In the absence of TRAP binding (i.e. when TRAP is not bound by excess tryptophan), an antiterminator structure forms that precludes formation of the terminator structure, and transcription proceeds into the trp structural genes (Fig. 1A).

For the trp system to be regulated by TRAP, sufficient free TRAP complex needs to be present to bind to newly made trp leader RNA, which is constitutively transcribed from the trp promoter. We found recently that the TRAP complex is released from trp leader RNA by rapid degradation of trp leader RNA (4). This degradation is accomplished by PNPase, whereas other 3'-to-5' exoribonucleases cannot digest RNA that is TRAP-bound, resulting in accumulation in a pnpA strain of an RNA fragment that contains trp leader nt 1–95. Thus, in a pnpA mutant, release of the TRAP complex from trp leader RNA is inefficient, leading to an insufficient amount of free TRAP to regulate trp transcription and an overexpression of trp operon structural genes.

The small size of trp leader RNA makes it an ideal model to study RNA turnover. (In this report, "trp leader RNA" refers to the 139-nt RNA that is the result of transcription termination in the trp leader sequence.) trp leader RNA is rapidly degraded in a wild-type strain with a half-life of about 1 min, significantly shorter than the average half-life of B. subtilis mRNAs (7). The presence of stem-loop structures at the 5' and 3' ends of trp leader RNA (Fig. 1B), as well as the binding of a large portion of the internal sequence by the TRAP complex, makes it particularly suitable as an object of study, as one would predict that there are few available internal sites in this molecule at which decay could initiate. Because it was known from our previous study that PNPase is responsible for trp leader RNA degradation, we studied PNPase activity on trp leader RNA in vitro. These experiments led to a hypothesis concerning initiation of trp leader RNA turnover in vivo, which was confirmed by analysis of decay of mutant trp leader RNA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains—The B. subtilis strain designated BG1, which is trpC2 thr-5, was the source of genomic DNA for cloning the PNPase coding sequence and the trp leader region. BG1 was also the host for experiments in vivo. A deletion strain that was missing the trp leader region was constructed by transformation of BG1 with plasmid pGD12 (see below) and integration at the trp locus with selection for spectinomycin resistance (200 µg/ml).

E. coli strain DH5{alpha} (8) was the host for construction of pGD5. XL1-Blue cells (9) were used for construction of pNP21 and pGD7. BL21(DE3)plysS (Invitrogen) carrying pNP21, designated EG763, was used for overexpression of His-tagged PNPase. A control strain, EG764, carried the pET15b vector alone. The host for construction of pGD12 was the StrataClone SoloPack strain (Stratagene).


Figure 1
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  		FIGURE 1.
Diagrams of trp leader RNA used for in vitro experiments. A, nucleotide sequence and predicted structure of trp leader RNA when the cellular concentration of tryptophan is low and the TRAP complex does not bind. Under these conditions, transcription proceeds into the trp operon. The 5' stem-loop (SL) structure and antiterminator structure are indicated. Numbering is from the start of in vivo transcription. B, trp leader RNA under conditions of high intracellular tryptophan, with the TRAP complex bound. The 11 trinucleotide repeats (UAG or GAG), each of which binds a TRAP monomer, are in bold. The GGG sequence near the 3' end that was mutated to CCC is indicated.

 
Plasmids—For overexpression of B. subtilis PNPase, the pnpA coding sequence was amplified using PCR primers that contained NdeI and BamHI sites. The PCR fragment was cloned into pET15b (Novagen), giving plasmid pNP21. For cloning of the trp leader, a 340-base pair fragment, starting from codon 84 of the aroH coding sequence until 53 bp downstream of the trp leader transcription terminator sequence, was amplified using PCR primers that contained SphI and EcoRI sites, and the PCR fragment was cloned into pSI45 (10) yielding pGD5. Plasmid pGD5 also contained the mtrAB operon (mtrB encodes TRAP). To mutagenize the trp leader RNA endonuclease cleavage site, two PCR fragments were generated. One fragment, which included the last 120 bp of the aroH coding sequence, was amplified with PCR primers containing an SphI site and a NotI site (the NotI site constituted the changed nucleotides at the trp leader RNA cleavage site). The second fragment, which included the first 80 bp of the trpE coding sequence, was amplified with PCR primers containing an EcoRI site and a NotI site. These fragments were annealed and amplified using the SphI- and EcoRI-containing primers, and the resulting PCR fragment was used to replace the wild-type trp leader DNA fragment of pGD5, giving pGD7.

For experiments in which mutant trp leader RNA was analyzed in vivo, the wild-type trp leader region of the host was deleted. For this, plasmid pGD12 was constructed. Three PCR fragments were prepared. The first PCR fragment contained the final 460 bp of the aroB coding sequence and extended 190 bp into the aroH coding sequence. The downstream primer included 52 nucleotides that were complementary to the 5' end of the second PCR fragment. The second PCR fragment contained the spectinomycin-resistance gene that is carried on plasmid pJL62 (11). The third PCR fragment contained bp 120–816 of the trpE coding sequence and included 29 nucleotides that were complementary to the 3' end of the second PCR fragment. These three fragments were annealed, amplified, and the resulting 2.7-kbp fragment was cloned into the pSC-A vector (Stratagene) giving plasmid pGD12. (It should be noted that this cloning could only be achieved when transformed cells were grown at 30 °C rather than 37 °C. This region of the chromosome seems to be detrimental to E. coli when present in high copy.) pGD12 DNA was linearized with BstBI and used to transform BG1. The resulting strain, which had a substitution of a spectinomycin-resistance gene for the trp leader region, was used in the experiments shown in Fig. 6.

Purification of PNPase—E. coli strains EG763 and EG764 were grown overnight at 37 °C in 2x YT medium (1% yeast extract, 2% tryptone, 1% NaCl) containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol. 10 ml of the overnight culture was added to 1 liter of 2x YT medium, and the culture was grown to an A600 of 0.6. PNPase expression was induced by addition of isopropyl 1-thio-beta-D-galactopyranoside to 1 mM followed by 30 min further incubation. His-tagged protein was isolated as described (12) using a HisTrap HP column (Amersham Biosciences). The His tag was removed from 1 mg of protein by thrombin cleavage followed by removal of thrombin and thrombin cleavage product according to standard protocols (Novagen). Protein was applied to a HisTrap HP column a second time, and flow-through fractions were collected, concentrated, and assayed for purity and phosphorolytic activity. Purified PNPase was stored at –20 °C as a 1 mg/ml stock in 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM dithiothreitol, and 50% glycerol. To confirm that the His tag had been completely removed, aliquots of purified PNPase were probed by Western blotting using an anti-His6 antibody. The pET15b-containing strain EG764 was treated in parallel, and no PNPase activity was detectable.

Assay of PNPase Activities—Uniformly labeled trp leader RNA was synthesized using T7 RNA polymerase transcription (Ambion T7 MAXIscript kit) in the presence of [{alpha}-32P]UTP of a PCR fragment that contained the trp leader sequence, with a T7 promoter included in the upstream primer. The transcription product was purified from a 6% denaturing polyacrylamide gel as described (13) using a diffusion buffer that contained 1 M ammonium acetate, 2 mM magnesium acetate, 1 mM EDTA, and 0.2% SDS. Degradation of uniformly labeled RNA substrates to dinucleotides was assayed by thin-layer chromatography developed in 1 M LiCl. Degradation reactions contained 0.5 nM labeled RNA substrate and 2.5 nM purified PNPase and were done in a buffer containing 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, and 2.5% glycerol, with or without 1 mM MgCl2 and 2 mM sodium phosphate. Incubation was for 10 min at 37 °C. The reactions were stopped by the addition of EDTA to 5 mM.


Figure 2
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  		FIGURE 2.
PNPase purification and binding. A, purified B. subtilis PNPase from an E. coli overexpressing strain after binding to the first HisTrap HP column (lane 1) and after thrombin cleavage and passage through the second HisTrap HP column (lane 2). B, thin-layer chromatography analysis of PNPase digestion products. Migration of digestion products is in the direction of the arrow. Migration of unlabeled monophosphate and diphosphate nucleosides is indicated on the left. C and D, band shift assay for PNPase binding of trp leader RNA in the absence (C) and presence (D) of TRAP. Migration of unbound and bound RNA is indicated at the left. Increasing concentrations of PNPase added are indicated at the top. Lanes with PNPase in panel C contained 1, 2, 3, 4, 5, 6, 8, 10, and 12.5 nM enzyme. Lanes with PNPase in panel D contained 1, 2, 3, 4, 6, 8, 10, and 12.5 nM enzyme.

 
For PNPase binding and degradation assays, 5'-end-labeled trp leader RNA was prepared. Unlabeled (~10 µg) trp leader RNA was synthesized using the Ambion T7 MEGAscript kit and was purified from a gel as described above. The 5' end of the RNA was dephosphorylated using calf intestine alkaline phosphatase (New England Biolabs) and then labeled using polynucleotide kinase (New England Biolabs) and [{gamma}-32P]ATP. Labeled RNA product was gel-purified. In binding reactions, 0.45 nM unlabeled trp leader RNA and 0.05 nM labeled trp leader RNA were incubated at 37 °C with different PNPase concentrations in 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 2.5% glycerol, 0.2 mM EDTA, and 2.5 mM tryptophan. In PNPase binding experiments with TRAP present, trp leader RNA was preincubated for 15 min at room temperature with 37.5 nM TRAP. To visualize migration of samples on native 10% polyacrylamide gels as described (14), 0.001% xylene cyanol was included during the reaction; this did not interfere with binding. Xylene cyanol was also present in the degradation assays (see below).

For PNPase degradation reactions, binding was allowed to occur as described above, and the phosphorolysis reaction was initiated by the addition of MgCl2 to 1 mM and sodium phosphate to 2 mM. The degradation reactions (200 µl) contained 0.45 nM unlabeled trp leader RNA, 0.05 nM 5'-end-labeled trp leader RNA, and 2.5 nM PNPase. 20-µl aliquots were removed at different time points, and the reaction was stopped by the addition of EDTA to 5 mM. Samples were put immediately on dry ice. An equal volume of Ambion gel loading buffer was added, and samples were loaded on a 6% denaturing polyacrylamide gel.

Quantitation of band shifts and decay products was done with a Storm 860 PhosphorImager instrument (GE Healthcare). For band shifts, the fraction of RNA bound was measured by the disappearance of unbound RNA (where TRAP was not present) or of RNA-TRAP complex (where TRAP was present). To determine the apparent dissociation constant, Kapp, theoretical curves were generated according to the formula: RNAbound = (R + P + Kapp)/(2 x R) – ([R + P + Kapp]2 – 4 x P x R)0.5/(2 x R) (where P = PNPase concentration, R = RNA concentration). The Kapp value was determined from the best fit of the theoretical curve to the experimental data. Fitting was done using the Solver feature of Microsoft Excel.

Northern Blot Analysis—RNA was isolated from B. subtilis strains as described (4). Measurement of trp leader RNA half-life after the addition of rifampicin to 150 µg/ml was as described (4). To control for RNA loading in Northern blot analyses, membranes were stripped and probed for 5 S rRNA as described (15). The size markers were end-labeled fragments of TaqI-digested plasmid pSE420 (16). 5'-end-labeled oligonucleotides were used as probes. Quantitation of mRNA half-life was determined by a linear regression analysis of percent RNA remaining versus time.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The B. subtilis pnpA coding sequence was cloned in the pET15b vector for overexpression in E. coli, yielding PNPase protein containing an N-terminal His tag and thrombin cleavage site (see "Experimental Procedures"). The tag was cleaved, and purified B. subtilis PNPase was judged >95% pure by Coomassie R staining (Fig. 2A). To test for phosphate-dependent activity of the purified enzyme, uniformly labeled trp leader RNA was incubated with PNPase with or without Mg2+ and inorganic phosphate. The products were analyzed by thin-layer chromatography and are shown in Fig. 2B. Degradation of the substrate to diphosphate nucleosides was dependent on the presence of Mg2+ and Pi. A slight amount of product was observed in the presence of Mg2+ without added Pi, which was likely due to trace amounts of Pi in the reaction buffer components. This small amount of activity, however, was less than 4% of the activity with Pi present and thus did not interfere with subsequent analyses.

A binding assay was developed in which the binding of PNPase to trp leader RNA could be assessed quantitatively. The wild-type trp leader RNA for these experiments consisted of the 139 nt of the trp leader sequence, with an additional three G residues at the 5' end to enhance in vitro transcription by T7 RNA polymerase. This RNA was called "trp RNA." (The additional three G residues are not shown in Fig. 1, to maintain the conventional trp leader numbering.) trp RNA was labeled at the 5' end and tested for binding by PNPase with increasing concentrations of the enzyme (Fig. 2C). The binding assay was done in the absence of Mg2+ and Pi to prevent degradation of the substrate. With increasing concentrations of PNPase, increasing amounts of the trp RNA shifted to slower migrating species, indicating PNPase binding (Fig. 2C). The multiple bands that were observed upon PNPase binding were due to the mixture of PNPase forms that were present in the purified preparation, as seen in a nondenaturing protein gel (data not shown). Although the active form of PNPase in the cell is a trimer (17), monomer, dimer, and trimer forms could be observed in vitro. For the apparent dissociation constant (Kapp) calculations, we assumed that each of these forms bind RNA with approximately the same affinity. A Kapp for PNPase binding was determined by measuring the disappearance of the trp RNA band, which was 1.0 ± 0.2 nM (Fig. 3A).


Figure 3
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  		FIGURE 3.
Plots of band shift data for trp RNA (A), trp RNA with the GGG -> CCC change in the terminator stem (B), and trp leader RNA with a poly(A) tail (C). Data are the average of 4 experiments ± S.D. The lines drawn are the theoretical curves calculated as described under "Experimental Procedures." Dashed lines, without TRAP; solid lines, with TRAP. To the right of each plot are schematic diagrams showing the predicted structure of the trp leader RNA with and without TRAP bound, as well as the respective Kapp values.

 
The addition of TRAP to the PNPase binding assay resulted in the appearance of a TRAP-bound species in addition to higher forms containing TRAP and PNPase (Fig. 2D). For the binding experiments that included TRAP, a concentration of TRAP was used (37.5 nM) that was determined to be saturating for binding under our assay conditions. In repeated experiments (not shown), the migration of the bound RNA was slightly slower when PNPase and TRAP were present than when PNPase alone was present. This indicates that both PNPase and TRAP can bind to the same trp leader RNA molecule. A Kapp for PNPase binding when TRAP was prebound was determined by measuring the disappearance of the TRAP-trp RNA complex. The calculated Kapp in this case was 9.7 ± 0.1 nM (Fig. 3A).

This large difference in Kapp with and without TRAP present was likely because of the 3'-proximal terminator structure that formed when TRAP was bound, which would inhibit binding of PNPase at the 3' end because there would be only 6 unpaired nt at the 3' end. To confirm this hypothesis, a mutant form of trp RNA was synthesized in which the three G residues near the 3' end (nt 133–135 in Fig. 1B) were changed to C residues, giving "trp CCC RNA." This change was predicted to drastically reduce the free energy of the 3'-terminal structure (including 3 nt on either side of the predicted stem-loop) from a {Delta}G0 of –13.0 kcal mol–1 for wild-type trp leader RNA to a {Delta}G0 of –2.2 kcal mol–1 for trp CCC RNA. Indeed, the Kapp for PNPase binding to trp CCC RNA was similar in the presence or absence of TRAP (Fig. 3B). These results demonstrated that the effect of TRAP on PNPase binding was not due to the presence of TRAP per se but was an indirect effect caused by formation of the terminator structure.

In addition to binding assays, degradation assays were performed. In these assays, the RNA-protein complex (RNA + PNPase or RNA + TRAP + PNPase) was allowed to form followed by the addition of Mg2+ and Pi to activate PNPase exonuclease activity. The data in Fig. 4A show that, in the absence of TRAP, degradation of trp RNA proceeded rapidly, with over 90% of the full-length RNA degraded in less than 5 min. In the presence of TRAP, however, decay was much slower. About 70% of the full-length RNA was still present even after 15 min incubation. The results of the degradation assay on trp RNA in the presence of TRAP reflected those of the binding assay. When TRAP was present, only about 60% of trp RNA was bound by PNPase even after extended incubation times (Fig. 3A). Thus, the capacity of PNPase to degrade trp RNA when it was bound by TRAP appeared to reflect the capacity to bind substrate, which was affected by the presence of the terminator structure. This was confirmed by a degradation assay of the trp CCC RNA, which could not form a stable terminator structure. PNPase could fully and rapidly degrade the trp CCC RNA even in the presence of TRAP (Fig. 4B).

The results thus far suggested that PNPase, acting alone, was incapable of rapidly degrading trp leader RNA. We considered the possibility that rapid decay of trp leader RNA in vivo was made possible by the addition of a poly(A) tail. A substrate that had 17 A residues at the 3' end, termed "trp (A)17 RNA," was prepared and tested for binding and decay by PNPase. Unlike the case of trp RNA, binding of PNPase to trp (A)17 RNA was unaffected by the presence of TRAP (Fig. 3C). The Kapp for PNPase binding was calculated to be 0.6 ± 0.2 nM in the absence of TRAP and 0.7 ± 0.1 nM in the presence of TRAP. Clearly, relocation of the 3' end distal to the transcription terminator structure allowed efficient PNPase binding. On the other hand, the degradation assay (Fig. 4C) showed that although PNPase could bind trp (A)17 RNA well, it could not degrade this substrate past the terminator structure that was formed when TRAP was present. A processing product of ~142 nt appeared shortly after initiation of the decay reaction, and this product represented a large fraction (60%) of the total full-length RNA. This product was consistent with a block to PNPase at the base of the terminator structure. When the substrate with the (A)17 tail was provided with the GGG -> CCC change in the terminator structure, to give "trp CCC (A)17 RNA," no processing product was observed (Fig. 4D). These results demonstrated that PNPase was incapable of completely degrading a substrate that had a strong terminator structure, even if a poly(A) tail was provided. The indication was that rapid decay of trp leader RNA in vivo could not be effected by attack from the native 3' end.


Figure 4
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  		FIGURE 4.
Degradation assays for various trp leader RNA substrates, incubated in the absence (–) or presence (+) of TRAP. The substrates are indicated above each set of gels. The marker lane (M) contained end-labeled DNA fragments. The marker fragment running just below trp RNA was 136 nt. Nine lanes after the marker lane in each gel contained an aliquot of the degradation assay, removed at the start of the reaction (first lane) and at subsequent times thereafter (1, 2, 4, 6, 8, 10, 12, and 16 min). To the right of each set of gels in A and B are plots of the fraction of RNA remaining versus time: closed squares, with TRAP; open squares without TRAP. For C and D, two plots are shown, one showing the fraction of full-length (FL) RNA remaining and the other the amount of 142-nt processed product accumulating. Migration of the full-length RNA (159 nt) and processed product (~142 nt) are indicated to the left of the lower gel in C.

 
A likely explanation for the ability of PNPase to rapidly degrade trp leader RNA in vivo was that exonucleolytic decay was preceded by endonuclease cleavage upstream of the terminator structure. If so, it should be possible to detect the products of such cleavage. We hypothesized that a reasonable location for such endonucleolytic cleavage would be in the region between the TRAP binding site and the terminator structure (Fig. 5A). Cleavage in this region would generate an upstream fragment of about 100 nt, which would be degraded by PNPase, and a downstream fragment of about 40 nt.

Northern blot analysis of trp leader RNA in a wild-type strain, grown in the presence of tryptophan, hinted at such an endonucleolytic cleavage (data not shown), but the level of trp leader RNA in this strain was too low to reliably detect decay intermediates. Therefore, a high-copy plasmid, pGD5, was constructed that contained the trp promoter and leader region sequence, as well as the TRAP-encoding gene to provide sufficient TRAP complex (see "Experimental Procedures"). Total RNA was isolated from the pGD5-containing strain that had been grown in the presence of tryptophan. Northern blot analysis of this RNA was performed using an "upstream probe" that was complementary to nt 74–99 of the trp leader RNA and a "downstream probe" that was complementary to nt 99–125 (Fig. 5A). A strong full-length RNA band (139 nt) was detected by both probes (Fig. 5, B and C). The upstream probe detected, in addition, a band that was ~100 nt; the downstream probe detected a group of bands centered at ~40 nt. The sizes of these bands were confirmed by Northern blot analysis of these samples run on a high-resolution (sequencing) gel (data not shown). The fact that the sum of the sizes of the different bands detected by the two probes added up to the size of the full-length trp leader RNA suggested strongly that endonucleolytic cleavage was occurring in the region between the TRAP binding site and the terminator structure.

The single-stranded region between the TRAP binding site and the terminator stem-loop structure (i.e. nt 93–107) is AU-rich, containing the sequence: CAUUAUGUUUAUUCU (nt 100 is underlined). To test whether the AU-rich sequence of this region was at least partly responsible for recognition by the putative endonuclease, a mutant version of pGD5 was constructed that had this sequence changed to eliminate many of the A and U residues around nt 100 as follows: CAUGCGGCCGCUUCU (changed nucleotides are underlined; the changed sequence constitutes a NotI restriction endonuclease site). This gave plasmid pGD7. Northern blot analysis of this RNA (Fig. 6A) showed that the wild-type ~100-nt band detected by the upstream probe and the wild-type ~40-nt group of bands detected by the downstream probe were no longer observed for the mutant trp leader RNA. (The band running below full-length trp leader RNA and detected by the upstream probe was also present, to a lesser degree, in the strain carrying pGD5. This may represent an alternative endonuclease cleavage site that is not affected by the change in sequence.) This experiment provided strong evidence that an endonuclease recognizes this AU-rich sequence, perhaps in the context of the neighboring structural elements.


Figure 5
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  		FIGURE 5.
Northern blot analysis of trp leader RNA processing in vivo. A, schematic diagram showing TRAP-bound trp leader RNA and extent of complementarity of the upstream and downstream 5'-end-labeled (asterisks) oligonucleotide probes. The dotted line indicates the AU-rich sequence that was mutated to give a NotI site in pGD7. B and C, Northern blot analysis of steady-state trp leader RNA using the two probes. Migration of full-length (FL) terminated RNA and processed fragments is indicated. The marker lanes contained end-labeled DNA fragments of the sizes indicated to the left.

 
If our hypothesis that trp leader RNA decay initiates with endonuclease cleavage at around nt 100 was correct, then we predicted that mutagenesis of the endonuclease target site would result in increased trp leader RNA half-life. To test this, Northern blot analysis of decay of wild-type and mutant trp leader RNA encoded by pGD5 and pGD7, respectively, was performed using the upstream oligonucleotide probe. The results (Fig. 6, B and C) showed clearly that, indeed, elimination of the endonuclease recognition site had resulted in a dramatic increase in mRNA half-life. Quantitation of the results (average of three experiments) showed that the wild-type trp leader RNA half-life was 2.7 ± 0.3 min and the mutant trp leader RNA half-life was 10.2 ± 1.6 min.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here are the first binding studies of a purified intracellular exoribonuclease from B. subtilis. Rather than focusing on the biochemical properties of B. subtilis PNPase, which, given the high sequence conservation of PNPases, are likely to be similar to others that have been studied in detail (17), the goal here was to determine the mechanism for rapid degradation of trp leader RNA, inasmuch as such rapid decay is a requirement of trp operon regulation. Mackie and colleagues (18) determined that the Kapp for binding of E. coli PNPase to an unstructured 3' end was 1.4 nM. Similarly, we determined a Kapp for binding of B. subtilis PNPase to trp leader RNA, in the absence of TRAP, to be 1.0 nM (Fig. 3A). The Kapp value for PNPase binding to trp leader RNA increased almost 10-fold when TRAP was present, and we demonstrated that this was due to formation of the 3' terminator structure. We believe this is the first time that the quantitative effect of 3'-terminal secondary structure on PNPase binding has been determined. Although the addition of a poly(A) tail to E. coli RNAs has been shown to increase lability in vivo (19), we observed in vitro that addition of a poly(A) tail resulted only in an increase in binding but did not aid in the turnover of upstream trp leader RNA, which was TRAP-bound (Fig. 4C). This result suggests that provision of a poly(A) tail does not simply provide a "toehold" for PNPase to bind, after which PNPase can digest through strong secondary structure. Rather, as has been suggested (20), it is likely that an iterative process of repeated poly(A) addition and processive exonuclease activity is required to turn over RNAs that have a 3'-proximal structure, such as a Rho-independent transcription terminator. We note that the gene encoding poly(A) polymerase of B. subtilis has not yet been identified. Experiments that can determine the effect of poly(A) addition on RNA turnover in B. subtilis await the identification of the poly(A) polymerase gene and construction of mutant strains lacking such an activity.


Figure 6
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  		FIGURE 6.
Northern blot analysis of wild-type and mutant trp leader RNA processing in vivo. A, comparison of steady-state pattern of wild-type (wt) and mutant (mut) trp leader RNAs carried on pGD5 and pGD7, respectively, using upstream and downstream oligonucleotide probes as indicated. Bands migrating above full-length terminated trp leader RNA (FL), which are not marked in the figure, were detectable by an oligonucleotide probe that was complementary to sequences downstream of the trp leader RNA terminator (data not shown). Thus they represent transcriptional read-through products. M, marker lane. B, Northern blot analysis of decay of wild-type and mutant trp leader RNA. The numbers above each lane indicate time (minutes) after rifampicin addition. The arrow points to full-length terminated trp leader RNA. C, plotting of half-life data, representing an average of three experiments. Closed diamonds, wild type; open diamonds, mutant.

 
E. coli PNPase can be found in the degradosome, which also includes RNase E and RhlB, an RNA helicase. Association with an RNA helicase activity could render 3'-terminal stem structures sensitive to PNPase digestion. For B. subtilis, however, only one of the putative four helicases, encoded by the deaD gene, is known to have RNA-helicase activity, and this is specifically activated by 23 S rRNA (21). Also, there is no evidence as yet for a degradosome-like complex in B. subtilis (22).

Mutagenesis of trp leader RNA at the endonuclease cleavage site resulted in a 4-fold increase in RNA half-life. Previously, we had determined the half-life of trp leader RNA to be 1.3 min (4). The observed half-life of wild-type trp leader RNA here was about twice as long (2.7 min). This difference may be attributed to the fact that here we were not measuring decay of native trp leader RNA encoded on the chromosome but, rather, decay of trp leader RNA encoded on the high-copy plasmid pGD5. Although the plasmid also contained the mtrB gene, encoding TRAP, it is possible that the ratio of the TRAP complex to trp leader RNA in the strain carrying pGD5 is different from the ratio in the native situation, such that the half-life of trp leader RNA is affected. In any event, stabilization of trp leader RNA by mutation of a single site is a noteworthy result. This showed directly that the short, single-stranded region between the TRAP binding site and the transcription terminator structure is a target for decay-initiating endonuclease cleavage. The small size of the trp leader RNA, the presence of 5' and 3' double-stranded structure, and the presence of TRAP bound to a major internal portion of the RNA likely render this RNA susceptible to endonuclease cleavage at only a single site. This is the first time that such a site has been mapped in B. subtilis. In similar experiments with small RNAs in E. coli, it was shown that mutagenesis of a single RNase E or RNase III cleavage site can have profound effects on mRNA decay (23–26). The endonuclease required for rapid trp leader RNA turnover may be the 5'-binding endonuclease, which is suggested by genetic experiments to be responsible generally for initiation of mRNA decay in B. subtilis (22). It is anticipated that use of the trp leader RNA as a substrate in vivo and in vitro will be useful in identifying this endonuclease.


   FOOTNOTES
 
* This work was supported by United States Public Health Service Grant GM48804 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Box 1603, 1 Gustave L. Levy Place, New York, NY 10029-6574. Tel.: 212-241-5628; Fax: 212-996-7214; E-mail: david.bechhofer{at}mssm.edu.

2 The abbreviations used are: PNPase, polynucleotide phosphorylase; nt, nucleotide(s); TRAP, trp RNA-binding attenuation protein. Back


   ACKNOWLEDGMENTS
 
We thank Paul Babitzke for purified TRAP.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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