ABSTRACT
Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation.
IMPORTANCE CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity.
INTRODUCTION
Bacteria sense and respond to environmental signals through the use of a variety of global regulatory networks, resulting in sweeping changes in gene expression. The Csr system is one such network that globally controls gene expression posttranscriptionally (reviewed in references 1, 2, and 3). Depending on the organism, Csr regulates a variety of cellular processes, including virulence, motility, quorum sensing, biofilm development, and carbon metabolism. CsrA is an RNA binding protein and the central component of the Csr system. Homodimeric CsrA contains two identical RNA binding surfaces and is capable of simultaneously binding two sites within a target transcript (4–6). GGA is a highly conserved motif in CsrA binding sites, and this sequence is often present in the loop of RNA hairpins (7, 8).
Binding of Escherichia coli CsrA to target transcripts represses or activates gene expression by regulating translation initiation and by altering the stability of target transcripts and, in one instance, was shown to promote Rho-dependent termination (9–12). Complex regulatory circuitry tightly controls the activity of CsrA in the cell. Multiple σ70- and σS-dependent promoters drive transcription of csrA, while CsrA directly represses its own translation (13). Two small RNA (sRNA) antagonists, CsrB and CsrC, in turn, control CsrA activity; these sRNAs contain several CsrA binding sites and are thus capable of sequestering multiple CsrA dimers (7, 14). The BarA-UvrY two-component signal transduction system activates transcription of csrB and csrC in response to the presence of short-chain fatty acids, particularly acetate (15, 16), whereas CsrD is a protein that targets CsrB and CsrC for degradation by RNase E and PNPase (17). Lastly, the DEAD box RNA helicase DeaD activates translation of UvrY (18). As transcriptome sequencing (RNA-seq) identified >700 transcripts that bind to CsrA, this protein may directly affect expression of ∼15% of the genes in E. coli (19). Moreover, since >40 of the transcripts identified by RNA-seq encode regulatory proteins, it is apparent that Csr directly or indirectly affects expression of a large fraction of the E. coli genome.
CsrA represses translation initiation by binding to as many as six sites in target transcripts (20). In the majority of these cases, one of the sites overlaps the Shine-Dalgarno (SD) sequence, such that bound CsrA directly competes with ribosome binding (4, 6, 10, 13, 19–22). PNPase, encoded by pnp, is a 3′-to-5′ exoribonuclease and a component of the E. coli degradosome, a protein complex involved in cellular RNA turnover (23, 24). PNPase participates in an autoregulatory mechanism that also involves RNase III, a double-strand-specific endoribonuclease. RNase III cleavage in the untranslated leader region of pnp mRNA, followed by PNPase-mediated exonucleolytic digestion of the resulting 5′ fragment, results in a pnp transcript that is somehow subject to translational repression and rapid degradation (25, 26). As pnp was identified as a CsrA target by RNA-seq (19), we explored the possibility that CsrA participates in this autoregulatory mechanism. Here we show that CsrA represses translation of pnp mRNA but only after the transcript is fully processed by RNase III and PNPase.
MATERIALS AND METHODS
Bacterial strains and plasmids.All bacterial strains used in this study are listed in Table 1. All numbering throughout the manuscript is with respect to the start of pnp translation. E. coli strain S17-1 λpir (27) was used for conditional-replication, integration, and modular (CRIM) plasmid construction (30). CRIM translational fusions using plasmid pLFT (19) were generated as follows. Plasmid pHP4 contains the pnp promoter, leader, and initially translated regions (positions −245 to +182 relative to the start of pnp translation) cloned between the PstI and BamHI sites of pLFT, thereby generating a pnp′-′lacZ translational fusion (where ′-′ indicates that pnp was truncated at the 3′ end and lacZ was truncated at the 5′ end). A GGA-to-GAG mutation in CsrA binding site 1 (BS1) was introduced using the QuikChange protocol (Stratagene), resulting in plasmid pHP11. This construct was generated so that base pairing in the stem was maintained (Fig. 1, left panel). The wild type (WT) and the mutant fusion were integrated into the chromosomal λ att site of strain CF7789 as described previously (30), resulting in strains PLB2176 and PLB2198, respectively. The csrA::kan (29) and pnpΔ683::(Str Spr) (28) alleles were introduced into PLB2176 by P1 transduction using TRMG1655 and SK10019 as donor strains, resulting in strains PLB2184 and PLB2415, respectively. Similarly, strain PLB2418 was constructed by transferring the pnpΔ683::(Str Spr) allele into PLB2184.
E. coli strains used in this study
Model of CsrA-mediated repression of pnp translation. CsrA is unable to bind to the pnp leader transcript prior to processing by RNase III and PNPase because the two CsrA binding sites (BS1 and BS2) are sequestered in an RNA secondary structure (left). A mutation in BS1 was introduced so as to maintain base pairing within the stem (inset). RNase III-mediated cleavage leaves a 2-nt 3′ extension on the upstream cleavage fragment, which is a substrate for PNPase (center). PNPase degrades the 5′ fragment, and, following a structural rearrangement, CsrA is able to bind to the two single-stranded binding sites and repress pnp translation (right). The highly conserved GGA motif of each CsrA binding site is highlighted. Positions of the Shine-Dalgarno (SD) sequence and translation initiation codon (Met) are also shown. RNA structural predictions were carried out with MFOLD (44) using constraints based on RNA structure-mapping experiments presented here.
The rnc::Cmr knockout PLB2409 strain was constructed by following a published procedure (31). Briefly, the pKD46 helper plasmid and a PCR product in which the first 210 codons of rnc were replaced with the cat gene from pKD3 were used to disrupt rnc while maintaining expression of the essential downstream era gene. The resulting rnc::Cmr allele was confirmed by PCR. The rnc::Cmr allele was subsequently transduced into strains PLB2176 and PLB2184, resulting in strains PLB2416 and PLB2419, respectively.
β-Galactosidase assay.Bacterial cultures containing pnp′-′lacZ translational fusions were grown in Luria-Bertani (LB) broth supplemented with 50 μg/ml ampicillin at 37°C and harvested at various times throughout growth. β-Galactosidase activity was determined as described previously (32). At least three independent experiments were performed for each strain.
Gel mobility shift assay.Quantitative gel mobility shift assays followed a published procedure (21). His-tagged CsrA was purified as described previously (33). RNAs containing the full-length pnp leader (pnpFl) or one that began at the 3′ RNase III cleavage site at position −81 (pnpPr) were synthesized using a RNAMaxx high-yield transcription kit (Agilent Technologies) and PCR-generated DNA templates. In addition, mutant pnpPr RNAs were generated in which the GGA motif in CsrA BS1 and/or BS2 was changed to CCA. Gel-purified RNA was dephosphorylated and then 5′ end labeled using T4 polynucleotide kinase (New England BioLabs) and [γ-32P]ATP (7,000 Ci/mmol). Labeled RNAs were renatured by heating for 1 min at 80°C and slow cooling to 25°C. Binding reaction mixtures (10 μl) contained 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol, 4 U of RNase inhibitor (Promega), 0.1 nM labeled RNA, purified CsrA-H6 (various concentrations), and 0.1 mg/ml xylene cyanol. Competition assays also contained unlabeled RNA competitors. Reaction mixtures were incubated for 30 min at 37°C to allow CsrA-RNA complex formation and then fractionated through 15% polyacrylamide gels. Free and bound RNA species were visualized with a Typhoon 9410 phosphorimager (GE Healthcare), and the apparent equilibrium binding constants (Kd) of CsrA-RNA interactions were calculated as described previously (11).
Enzymatic footprinting.Labeled RNAs were prepared as described above. Binding reaction mixtures (10 μl) were identical to those used in the gel mobility shift assay except that the concentration of labeled pnp RNA was increased to 50 nM and 200 μg/ml acetylated bovine serum albumin (BSA) was included in the reaction mixture. Following a 15-min incubation at 37°C to allow CsrA-RNA complex formation, RNase T1 (0.12 U) was added and incubation was continued for 15 min at 37°C. Reactions were stopped by addition of 10 μl of stop buffer (95% formamide, 0.025% SDS, 20 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol, 760 μg/ml yeast RNA), and the reaction mixtures were placed on ice. RNase T1 and base hydrolysis ladders were prepared as described previously (34). Samples were fractionated through a 6% (vol/vol) polyacrylamide–8 M urea sequencing gel. Cleavage products were visualized using a phosphorimager.
mRNA half-life analysis.Strains MG1655 (WT) and TRMG1655 (csrA::kan) (29) were grown at 37°C in LB broth to the exponential phase prior to the addition of rifampin (200 μg/ml) to prevent transcription initiation. After 1 min following rifampin addition, 0.8-ml aliquots were removed at various times and total cellular RNA was prepared by the hot phenol method as described previously (35). For Northern analysis, RNA samples were fractionated through 1.0% denaturing formaldehyde-agarose gels. RNA was transferred to a Hybond N+ membrane (Amersham Biosciences) by capillary blotting with 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (pH 7.0) for 16 h and fixed to the membrane by UV cross-linking at a wavelength of 302 nm for 3 min. Oligonucleotides pnpTL+11R (5′-GGATTAAGCAATGTAATATCCTTTCTC-3′) for pnp mRNA hybridization and 16SrRNAR (5′-GCAGGTTCCCCTACGGTTACCT-3′) for 16S rRNA hybridization, which served as a loading control, were 5′ end labeled with [γ-32P]ATP and T4 polynucleotide kinase (New England BioLabs). Hybridization was performed according to the manufacturer's instructions (Amersham Biosciences). Labeled RNA species were visualized with a phosphorimager and quantified using ImageJ software (36).
In vitro coupled transcription-translation assay.Plasmid pT7-pnpFl′-′lacZ contains a T7 promoter driving transcription of a pnp′-′lacZ translational fusion (−156 to +182). This plasmid was designed such that transcription initiated at the natural pnp transcription start site but with two additional G residues for initiation by T7 RNA polymerase (RNAP). Plasmid pT7-pnpPr′-′lacZ was constructed in a similar manner except that transcription began at −81, thereby mimicking a pnp transcript that was fully processed by RNase III and PNPase. Plasmid pT7-pnpPrM′-′lacZ is identical to pT7-pnpPr′-′lacZ except that it contains a GGA-to-GAG mutation in BS1. These three plasmids were used as the templates for coupled transcription-translation reactions using a PURExpress in vitro protein synthesis kit according to the instructions of the manufacturer (New England BioLabs). Reaction mixtures containing 20 nM plasmid DNA template and various concentrations of purified His-tagged CsrA were incubated for 2 h at 37°C. β-Galactosidase activity was determined according to the manufacturer's instructions.
RESULTS
CsrA represses pnp expression.PNPase is a 3′-to-5′ exoribonuclease and a component of the E. coli degradosome (23, 24). PNPase participates in an autoregulatory mechanism that also involves RNase III. RNase III-mediated cleavage of a pnp leader RNA structure removes the top portion of the stem such that the transcript containing the downstream pnp coding sequence remains base paired to the upstream (5′) cleavage fragment (Fig. 1). PNPase then degrades the 5′ fragment from the newly generated 3′ end, resulting in a pnp transcript that is regulated by an unknown translational repression mechanism (25, 26). Our previously published RNA-seq studies identified pnp as a CsrA target (19). Visual inspection of the untranslated leader region of pnp mRNA identified two putative CsrA binding sites, each containing the highly conserved GGA motif. The downstream site (BS2) overlaps the pnp SD sequence, suggesting that CsrA might be capable of repressing pnp translation. Prior to the action of RNase III and PNPase, the upstream site (BS1) would be sequestered in the large secondary structure, perhaps inhibiting CsrA binding (Fig. 1). Thus, we tested a model in which CsrA participates in the pnp regulatory mechanism by repressing translation following the action of RNase III and PNPase. Translational repression could then lead to accelerated decay of pnp mRNA.
We first examined expression of a pnp′-′lacZ translational fusion in wild-type and CsrA-deficient strains. Expression in the wild-type background was low throughout growth. In contrast, expression of the fusion in the csrA mutant background increased dramatically in exponential phase and remained high in stationary phase, indicating that csrA represses pnp expression throughout growth, with maximal 20-fold repression occurring during stationary phase (Fig. 2). We also examined expression of a fusion in which the GGA motif in BS1 was replaced with GAG. This mutant leader was designed to prevent CsrA binding to BS1 while maintaining base pairing of the long stem recognized by RNase III (Fig. 1, left panel). Expression of the mutant fusion was ∼5-fold higher than expression of the wild-type fusion, suggesting that CsrA is unable to fully repress pnp expression when CsrA is not bound to BS1. We did not test mutations in BS2 since mutations in this site would also disrupt the pnp SD sequence.
CsrA represses pnp expression. Expression of chromosomally integrated wild-type (WT) and BS1 mutant pnp′-′lacZ fusions was examined in WT or csrA mutant strains. β-Galactosidase activity (Miller units) ± standard deviation is shown with solid lines, while growth (optical density at 600 nm [OD600]) is shown with dashed lines. Experiments were performed at least three times.
CsrA binds to fully processed pnp mRNA.To determine whether processing of the pnp leader transcript by RNase III and PNPase affects CsrA binding, we performed quantitative gel mobility shift assays with two in vitro-generated transcripts, both of which contained BS1 and BS2. One transcript contained the full-length pnp leader (pnpFl), while the 5′ end of the other transcript began at the 3′ RNase III cleavage site (pnpPr), thereby mimicking the transcript produced in vivo following RNase III and PNPase processing (Fig. 3A). CsrA bound tightly to fully processed pnpPr RNA with an apparent Kd of 20 nM. In contrast, only weak binding to full-length pnpFl RNA was observed at the highest CsrA concentration tested (Fig. 3B and E). The specificity of CsrA-pnpPr RNA interaction was investigated by performing competition experiments with specific (pnpPr) and nonspecific (phoB) unlabeled RNA competitors. Unlabeled pnpPr was an effective competitor, whereas phoB was not, indicating that the CsrA-pnp RNA interaction is specific (Fig. 3C). We also tested CsrA binding to pnpPr RNA containing GGA-to-CCA mutations in BS1 and/or BS2 (Fig. 3D and E). CsrA bound to the transcript containing BS1 only (BS2 mutant) with an apparent Kd of 57 nM, while the affinity of CsrA for the transcript containing BS2 only (BS1 mutant) was about 2-fold lower. Binding was not observed to the transcript containing mutations in both binding sites.
Gel mobility shift analysis of CsrA-pnp leader RNA interaction. (A) Two 5′-end-labeled RNAs were tested that mimic the unprocessed full-length pnp leader (pnpFl) and the fully processed leader that results following RNase III cleavage and exonucleolytic degradation by PNPase (pnpPr) (Fig. 1). (B) Gel shift assay showing weak binding to pnpFl RNA and tight binding to pnpPr RNA. The nanomolar concentration of CsrA is shown at the top of each lane. Positions of bound (B) and free (F) RNA are also shown. (C) RNA competition experiment demonstrating binding specificity. Labeled pnpPr RNA was incubated with the indicated nanomolar concentration of CsrA ± a 10-fold or 100-fold excess of unlabeled specific (pnpPr) or nonspecific (phoB) competitor RNA (RNA comp). (D) Gel shift assay of CsrA binding to pnpPr RNA containing GGA-to-CCA mutations (mut) in BS1 and/or BS2. The nanomolar concentration of CsrA is shown at the top of each lane. (E) Simple binding curves for CsrA interaction with wild-type (WT) pnpPr RNA and the corresponding BS1 and BS2 mutant transcripts.
CsrA-pnpPr RNA footprint experiments were performed to verify CsrA binding to BS1 and BS2. Bound CsrA reduced RNase T1-mediated cleavage of G residues at positions −73, −71, −70, −67, and −65 within BS1. Similarly, CsrA prevented cleavage of positions −10 and −9 within BS2 (Fig. 4). In addition to protection of BS1 and BS2, bound CsrA caused increased cleavage of G3 within the UUG start codon and at positions G16 and G20 further downstream, indicating that CsrA binding alters the RNA structure in the translation initiation region. The finding that CsrA protected BS1 at the lowest concentration of CsrA used in this analysis, while protection of BS2 required a higher CsrA concentration, is consistent with our gel shift analysis demonstrating that CsrA has higher affinity for BS1 (Fig. 3D). In addition, our structure mapping data in the control lane without CsrA (Fig. 4) are consistent with the two CsrA binding sites being single stranded in the fully processed RNA, as depicted in our RNA structural model (Fig. 1, right panel). Taken together, our binding studies indicate that BS1 and BS2 constitute authentic CsrA binding sites. Moreover, our results demonstrate that CsrA binds to fully processed pnp mRNA with high affinity and specificity but not to the unprocessed pnp leader transcript.
CsrA-pnp leader RNA footprint analysis. (A) Sequence of pnp leader RNA. Positions of the downstream RNase III cleavage site, CsrA binding sites 1 (BS1) and 2 (BS2), Shine-Dalgarno (SD) sequence, translation start codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked. (B) CsrA-pnp leader RNA footprint analysis. 5′ end-labeled pnp leader RNA was treated with RNase T1 in the presence of the concentration of CsrA shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase treatment (C), are shown. Positions of BS1, BS2, the translation initiation codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked.
CsrA represses pnp translation.There are two mechanisms in which CsrA has been shown to repress gene expression posttranscriptionally. First, bound CsrA can decrease the stability of pnp mRNA. Second, CsrA can repress translation by blocking ribosome binding. These two possibilities are not mutually exclusive, as destabilization of mRNA can be an indirect consequence of translation inhibition. We first examined the stability of pnp mRNA in WT and CsrA-deficient strains by Northern blotting. The pnp half-life durations in the wild-type and csrA mutant strains were 4.3 and 6.3 min, respectively (Fig. 5). This difference is too small to account for the large difference in expression observed in the pnp′-′lacZ translational fusion studies (Fig. 2).
Northern blot analysis of pnp mRNA half-lives in wild-type (WT) and csrA mutant strains. Cultures were grown at 37°C to the mid-exponential phase prior to the addition of rifampin. After 1 min, samples were harvested at the indicated times. mRNA half-lives (T1/2) ± standard deviations are shown at the bottom of the gel. The level of pnp mRNA was normalized to the 16S rRNA level in each lane. Experiments were performed twice, and a representative gel is shown. Quantifications of these data are shown in the bottom panel.
We next used the in vitro coupled transcription-translation PURExpress system to determine whether CsrA inhibits pnp translation. Three different plasmids carrying pnp′-′lacZ translational fusions, all of which were driven by identical T7 RNA polymerase promoters, were used in this analysis. One plasmid gave rise to full-length pnp leader RNA (pnpFl) identical to that used in our in vivo expression studies (Fig. 2), while a second was designed to generate a transcript mimicking the fully processed pnp transcript (pnpPr). These two transcripts were essentially identical to those used in our gel shift analysis (Fig. 3A and B). The third construct contained the BS1 GGA-to-GAG mutation in pnpPr. Expression from each construct was measured by determining β-galactosidase activity. Addition of CsrA to the system caused a slight decrease in expression of the pnpFl construct; expression was reduced only 3% and 19% at 2.5 and 10 μM CsrA, respectively (Fig. 6). In contrast, expression of the pnpPr construct was reduced 54% and 91% at the same two CsrA concentrations. Expression of the pnpPr construct containing the BS1 mutation exhibited an intermediate level of CsrA-mediated repression. We conclude that bound CsrA represses translation of pnp but only after the RNA has been fully processed by RNase III and PNPase. These data also suggest that the small effect of CsrA on pnp mRNA stability (Fig. 5) was an indirect consequence of altered translational repression or RNA secondary structure.
Effect of CsrA on pnp translation. Coupled transcription-translation reactions were performed with a PURExpress kit using WT pnpFl, WT pnpPr, and BS1 mutant pnpPr DNA templates containing pnp′-′lacZ translational fusions. Purified CsrA protein was added prior to starting the reaction. β-Galactosidase activity normalized to 0 μM CsrA for each template is shown. Each experiment was performed at least twice, with representative results shown.
CsrA-mediated repression of pnp expression requires prior processing by RNase III and PNPase.Our data described above are consistent with a model in which CsrA represses translation of pnp transcripts following processing by RNase III and PNPase (Fig. 1). In the absence of processing by these two ribonucleases, the CsrA binding sites would be inaccessible, since both binding sites are sequestered in the RNA secondary structure. However, in the unprocessed transcript, the pnp SD sequence would be sequestered in an RNA structure, which would inhibit translation (Fig. 1, left panel). Following processing by these two nucleases, the pnp leader RNA refolds such that the SD sequence is single stranded and available for ribosome binding. However, this processed and refolded transcript would then be subject to CsrA-mediated translational repression since both of the CsrA binding sites are single stranded. Since our model predicts that CsrA functions downstream of RNase III and PNPase, we expected that disrupting rnc or pnp would suppress the effect of the csrA mutation on pnp expression. Thus, epistasis studies were carried out to test this model by examining expression of a pnp′-′lacZ translational fusion containing the entire leader region. Since processing of the pnp transcript is initiated by RNase III-mediated cleavage, we first compared expression levels of the pnp′-′lacZ fusion in WT, csrA, rnc, and csrA rnc strains (Fig. 7A). As we had previously observed (Fig. 2), pnp expression was much higher in the csrA mutant, which reflects the loss of CsrA-mediated translational repression of the fully processed transcript. We also found that expression was elevated in the rnc strain, indicating that sequestration of the pnp SD sequence in the unprocessed mRNA is less effective at repressing translation than CsrA-mediated repression of processed transcripts (Fig. 7A). Notably, as predicted by our model, the large increase in expression of the csrA mutant strain was completely suppressed in the RNase III-deficient background. However, we cannot account for the unexpected result that expression in the csrA rnc double-mutant strain was actually 2-fold lower than expression in the rnc strain.
Effects of CsrA, RNase III, and PNPase on pnp expression. β-Galactosidase activity (Miller units) ± standard deviations of a chromosomally integrated pnp′-′lacZ translational fusion is indicated (solid lines). A representative growth curve is shown for each strain (dashed lines). Each experiment was performed at least three times. (A) Expression in wild-type (WT), csrA, rnc, and csrA rnc strains. (B) Expression in WT, csrA, pnp, and csrA pnp strains.
We next compared expression levels in the WT, csrA, pnp, and csrA pnp strains (Fig. 7B). Expression was elevated in the pnp mutant strain but to a much smaller extent than in the csrA strain. Importantly, as predicted by our model, the large increase of expression in the csrA mutant strain was completely suppressed in the PNPase-deficient background; expression levels in the pnp and pnp csrA strains were essentially identical. Taken together with the results of our in vitro studies, we conclude that CsrA is effective only at binding to and repressing translation of pnp transcripts that are fully processed by RNase III and PNPase.
DISCUSSION
PNPase is one of three 3′ to 5′ exoribonucleases responsible for the degradation of bulk mRNA in E. coli (reviewed in reference 37). The other two enzymes, RNase II and RNase R, degrade RNA by a hydrolytic mechanism with the release of nucleoside monophosphates, whereas PNPase degrades RNA through a phosphorolytic mechanism, resulting in the release of nucleoside diphosphates. PNPase is also capable of catalyzing the reverse reaction, thereby synthesizing RNA from nucleoside diphosphates. This activity is responsible for the synthesis of polynucleotide tails on the 3′ ends of transcripts in strains lacking PAP I, the enzyme normally responsible for adding poly(A) tails; single-stranded 3′ tails provide a toehold for 3′ to 5′ exonucleases to degrade structured RNA (38). Although PNPase is capable of degrading RNA as a trimer of identical subunits, this enzyme also functions as a component of the degradosome. This multisubunit complex also contains the endoribonuclease RNase E, which serves as the scaffold for degradosome assembly, the DEAD box RNA helicase RhlB, and the glycolytic enzyme enolase (23, 24). At low temperatures, an alternative degradosome forms in which DeaD (CsdA) replaces RhlB (39). PNPase also exists as a complex containing the PNPase homotrimer and two subunits of RhlB (40). The unwinding activity of a DEAD box RNA helicase in each of these complexes probably enables PNPase to degrade structured RNA substrates (37).
pnp expression is repressed by a mechanism that was previously shown to involve RNase III-mediated cleavage of a large secondary structure that forms in the untranslated leader of the pnp transcript, resulting in a 5′ fragment that remains base paired to the downstream sequence (Fig. 1). This 5′ fragment contains a 2-nucleotide (2-nt) 3′ extension that serves as a substrate for exonucleolytic digestion by PNPase (25, 26). It was noted that, following the action of these two enzymes, the processed transcript was subject to an unidentified translation repression mechanism (25, 26) and/or accelerated degradation by RNase E (41). We found that CsrA binds to two sites in the pnp transcript that is fully processed by RNase III and PNPase (Fig. 3 and 4). The affinity of CsrA was highest for BS1, which is 61 nt upstream of BS2, which overlaps the SD sequence. It was previously shown that a single CsrA dimer is capable of bridging a high-affinity site to a lower-affinity site (6). Thus, it is apparent that dual-site bridging participates in the mechanism leading to dramatic repression of pnp translation (Fig. 2 and 6). In the absence of processing by RNase III and PNPase, the critical GGA motif of both CsrA binding sites remains sequestered in the RNA secondary structure (Fig. 1 and 4). Since CsrA binding requires that the GGA motif is unpaired (3, 5, 8), CsrA is unable to bind to unprocessed transcripts (Fig. 3 and 6). Indeed, the large increase in pnp expression observed in the csrA mutant is suppressed by mutations in either rnc or pnp, indicating that the ability of CsrA to repress pnp expression is eliminated in strains lacking RNase III or PNPase (Fig. 7). However, the finding that pnp expression was higher in the rnc strain than in the pnp strain (Fig. 7) indicates that our model does not fully account for the effects of these two enzymes. This is not surprising, as one would expect these globally acting nucleases to affect pnp expression by a variety of direct and indirect mechanisms. For example, a recent study found that PNPase directly represses its translation in an RNase III-independent manner (42).
CsrA-mediated repression of pnp translation was observed throughout growth, although the extent of the repression was most pronounced in stationary phase (Fig. 2 and 7). The increase in repression as cells exit exponential growth parallels the previously observed activation of csrA transcription mediated by RpoS (σS), the general stress response sigma factor of RNA polymerase (13). Under our growth conditions, the concomitant increases in CsrA levels and CsrA-mediated translational repression resulted in similar levels of pnp expression throughout growth (Fig. 2 and 7). This finding suggests that E. coli evolved an elaborate mechanism to maintain near-constant levels of PNPase in the cell. However, as CsrA activity is regulated by its two sRNA antagonists CsrB and CsrC (7, 14), alternative growth conditions or stresses leading to rapid changes in CsrB and CsrC levels could result in fluctuations in PNPase levels. For example, the Csr system reciprocally regulates the stringent response (19), which is characterized by a rapid downshift in ribosome biogenesis in response to amino acid starvation (reviewed in reference 43). Of particular interest, DksA and ppGpp, the mediators of the stringent response, activate csrB and csrC transcription 10-fold, whereas CsrA represses translation of relA, the gene encoding one of two ppGpp synthases. This complex circuitry suggests that CsrA-mediated regulation of gene expression is relieved during the stringent response (19). Thus, amino acid starvation, and perhaps other stress conditions that increase levels of CsrB and CsrC, is predicted to result in higher levels of PNPase.
ACKNOWLEDGMENTS
Hongmarn Park performed experiments, interpreted results, and wrote the manuscript. Helen Yakhnin performed experiments and interpreted results. Michael Connolly performed experiments and interpreted results. Tony Romeo designed the study, edited the manuscript, and secured funding. Paul Babitzke designed and supervised the study, wrote the manuscript, and secured funding.
We thank Louise McGibbon for critical reading of the manuscript and Sidney R. Kushner for providing strain SK10019 containing the pnpΔ683::(Str Spr) allele.
This work was supported by National Institutes of Health grant GM059969 to Tony Romeo and Paul Babitzke.
FOOTNOTES
- Received 28 August 2015.
- Accepted 28 September 2015.
- Accepted manuscript posted online 5 October 2015.
- Address correspondence to Paul Babitzke, pxb28{at}psu.edu.
↵* Present address: Michael Connolly, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.
Citation Park H, Yakhnin H, Connolly M, Romeo T, Babitzke P. 2015. CsrA participates in a PNPase autoregulatory mechanism by selectively repressing translation of pnp transcripts that have been previously processed by RNase III and PNPase. J Bacteriol 197:3751–3759. doi:10.1128/JB.00721-15.
REFERENCES
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