CsrA Participates in a PNPase Autoregulatory Mechanism bySelectively Repressing Translation of pnp Transcripts That Have BeenPreviously Processed by RNase III and PNPase

Hongmarn Park,a Helen Yakhnin,a Michael Connolly,a* Tony Romeo,b Paul Babitzkea

Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USAa; Departmentof Microbiology and Cell Science, University of Florida, Gainesville, Florida, USAb

ABSTRACT

Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichiacoli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability bybinding to the 5= untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3= to 5= exoribo-nuclease 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 IIIcleavage of the untranslated leader, followed by PNPase degradation of the resulting 5= fragment, leads to pnp repression by anundefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but onlyafter the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are seques-tered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site tothe downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnptranslation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrAhas been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first examplein 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 leaderregion preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leadertranscript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA second-ary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sitesoverlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation byCsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity.

Bacteria sense and respond to environmental signals throughthe use of a variety of global regulatory networks, resulting in

sweeping changes in gene expression. The Csr system is one suchnetwork that globally controls gene expression posttranscription-ally (reviewed in references 1, 2, and 3). Depending on the organ-ism, Csr regulates a variety of cellular processes, includingvirulence, motility, quorum sensing, biofilm development, andcarbon metabolism. CsrA is an RNA binding protein and the cen-tral component of the Csr system. Homodimeric CsrA containstwo identical RNA binding surfaces and is capable of simultane-ously binding two sites within a target transcript (4–6). GGA is ahighly conserved motif in CsrA binding sites, and this sequence isoften present in the loop of RNA hairpins (7, 8).

Binding of Escherichia coli CsrA to target transcripts repressesor activates gene expression by regulating translation initiationand by altering the stability of target transcripts and, in one in-stance, was shown to promote Rho-dependent termination (9–12). Complex regulatory circuitry tightly controls the activity ofCsrA in the cell. Multiple �70- and �S-dependent promoters drivetranscription of csrA, while CsrA directly represses its own trans-lation (13). Two small RNA (sRNA) antagonists, CsrB and CsrC,in turn, control CsrA activity; these sRNAs contain several CsrAbinding sites and are thus capable of sequestering multiple CsrAdimers (7, 14). The BarA-UvrY two-component signal transduc-

tion system activates transcription of csrB and csrC in response tothe presence of short-chain fatty acids, particularly acetate (15,16), whereas CsrD is a protein that targets CsrB and CsrC fordegradation by RNase E and PNPase (17). Lastly, the DEAD boxRNA helicase DeaD activates translation of UvrY (18). As tran-scriptome sequencing (RNA-seq) identified �700 transcripts thatbind to CsrA, this protein may directly affect expression of �15%of the genes in E. coli (19). Moreover, since �40 of the transcriptsidentified by RNA-seq encode regulatory proteins, it is apparent

Received 28 August 2015 Accepted 28 September 2015

Accepted manuscript posted online 5 October 2015

Citation Park H, Yakhnin H, Connolly M, Romeo T, Babitzke P. 2015. CsrAparticipates in a PNPase autoregulatory mechanism by selectively repressingtranslation of pnp transcripts that have been previously processed by RNase IIIand PNPase. J Bacteriol 197:3751–3759. doi:10.1128/JB.00721-15.

Editor: R. L. Gourse

Address correspondence to Paul Babitzke, pxb28@psu.edu.

* Present address: Michael Connolly, Temple University School of Medicine,Philadelphia, Pennsylvania, USA.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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that Csr directly or indirectly affects expression of a large fractionof the E. coli genome.

CsrA represses translation initiation by binding to as many assix sites in target transcripts (20). In the majority of these cases,one of the sites overlaps the Shine-Dalgarno (SD) sequence, suchthat bound CsrA directly competes with ribosome binding (4, 6,10, 13, 19–22). PNPase, encoded by pnp, is a 3=-to-5= exoribonu-clease and a component of the E. coli degradosome, a proteincomplex involved in cellular RNA turnover (23, 24). PNPase par-ticipates in an autoregulatory mechanism that also involves RNaseIII, a double-strand-specific endoribonuclease. RNase III cleavagein the untranslated leader region of pnp mRNA, followed byPNPase-mediated exonucleolytic digestion of the resulting 5= frag-ment, results in a pnp transcript that is somehow subject to transla-tional repression and rapid degradation (25, 26). As pnp was identi-fied as a CsrA target by RNA-seq (19), we explored the possibility thatCsrA participates in this autoregulatory mechanism. Here we showthat CsrA represses translation of pnp mRNA but only after the tran-script is fully processed by RNase III and PNPase.

MATERIALS AND METHODSBacterial strains and plasmids. All bacterial strains used in this study arelisted in Table 1. All numbering throughout the manuscript is with respectto the start of pnp translation. E. coli strain S17-1 �pir (27) was used forconditional-replication, integration, and modular (CRIM) plasmid con-struction (30). CRIM translational fusions using plasmid pLFT (19) weregenerated as follows. Plasmid pHP4 contains the pnp promoter, leader,and initially translated regions (positions �245 to �182 relative to thestart of pnp translation) cloned between the PstI and BamHI sites of pLFT,thereby generating a pnp=-=lacZ translational fusion (where =-= indicatesthat 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 introducedusing the QuikChange protocol (Stratagene), resulting in plasmid pHP11.This construct was generated so that base pairing in the stem was main-tained (Fig. 1, left panel). The wild type (WT) and the mutant fusion wereintegrated into the chromosomal � att site of strain CF7789 as describedpreviously (30), resulting in strains PLB2176 and PLB2198, respectively.The csrA::kan (29) and pnp�683::(Str Spr) (28) alleles were introducedinto PLB2176 by P1 transduction using TRMG1655 and SK10019 as do-nor strains, resulting in strains PLB2184 and PLB2415, respectively. Sim-ilarly, strain PLB2418 was constructed by transferring the pnp�683::(Str

Spr) allele into PLB2184.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 catgene from pKD3 were used to disrupt rnc while maintaining expression ofthe essential downstream era gene. The resulting rnc::Cmr allele was con-firmed by PCR. The rnc::Cmr allele was subsequently transduced intostrains PLB2176 and PLB2184, resulting in strains PLB2416 and PLB2419,respectively.

�-Galactosidase assay. Bacterial cultures containing pnp=-=lacZtranslational fusions were grown in Luria-Bertani (LB) broth supple-mented with 50 g/ml ampicillin at 37°C and harvested at various timesthroughout growth. -Galactosidase activity was determined as describedpreviously (32). At least three independent experiments were performedfor each strain.

Gel mobility shift assay. Quantitative gel mobility shift assays fol-lowed a published procedure (21). His-tagged CsrA was purified as de-scribed 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 BS1and/or BS2 was changed to CCA. Gel-purified RNA was dephosphory-lated and then 5= end labeled using T4 polynucleotide kinase (New Eng-land BioLabs) and [�-32P]ATP (7,000 Ci/mmol). Labeled RNAs were re-natured by heating for 1 min at 80°C and slow cooling to 25°C. Bindingreaction mixtures (10 l) contained 10 mM Tris-HCl (pH 7.5), 10 mMMgCl2, 100 mM KCl, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithio-threitol, 4 U of RNase inhibitor (Promega), 0.1 nM labeled RNA, purifiedCsrA-H6 (various concentrations), and 0.1 mg/ml xylene cyanol. Com-petition assays also contained unlabeled RNA competitors. Reaction mix-tures were incubated for 30 min at 37°C to allow CsrA-RNA complexformation and then fractionated through 15% polyacrylamide gels. Freeand bound RNA species were visualized with a Typhoon 9410 phosphor-imager (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 describedabove. Binding reaction mixtures (10 l) were identical to those used inthe gel mobility shift assay except that the concentration of labeled pnpRNA was increased to 50 nM and 200 g/ml acetylated bovine serumalbumin (BSA) was included in the reaction mixture. Following a 15-minincubation 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 l of stop buffer (95% formam-ide, 0.025% SDS, 20 mM EDTA, 0.025% bromophenol blue, 0.025% xy-lene cyanol, 760 g/ml yeast RNA), and the reaction mixtures were placedon ice. RNase T1 and base hydrolysis ladders were prepared as describedpreviously (34). Samples were fractionated through a 6% (vol/vol) poly-

TABLE 1 E. coli strains used in this study

Strain Descriptiona Source or reference

CF7789 �lacI-lacZ (MluI) M. CashelMG1655 Prototrophic M. CashelPLB2176 CF7789 pnp=-=lacZ Apr This studyPLB2184 CF7789 pnp=-=lacZ Apr csrA::kan This studyPLB2198 CF7789 pnp=-=lacZ (BS1 GGA-to-GAG mutation) Apr This studyPLB2409 CF7789 rnc::Cmr This studyPLB2415 CF7789 pnp=-=lacZ Apr pnp�683::(Str Spr) This studyPLB2416 CF7789 pnp=-=lacZ Apr rnc::Cmr This studyPLB2418 CF7789 pnp=-=lacZ Apr csrA::kan pnp�683::(Str Spr) This studyPLB2419 CF7789 pnp=-=lacZ Apr csrA::kan rnc::Cmr This studyS17-1 �pir recA thi pro hsdR17(rK

� mK�) RP4-2-Tc::Mu-Km::Tn7 �pir� 27

SK10019 thyA715 rph-1 pnp�683::(Str Spr) 28TRMG1655 MG1655 csrA::kan 29a The pnp=-=lacZ translational fusions were integrated into the � att site via the CRIM system (30). The pnp=-=lacZ fusion contained positions �245 to � 182 relative to the start ofpnp translation, including the natural pnp promoter and leader region. Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Sp, spectinomycin; St, streptomycin.

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acrylamide– 8 M urea sequencing gel. Cleavage products were visualizedusing 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 phaseprior to the addition of rifampin (200 g/ml) to prevent transcriptioninitiation. After 1 min following rifampin addition, 0.8-ml aliquots wereremoved at various times and total cellular RNA was prepared by the hotphenol method as described previously (35). For Northern analysis, RNAsamples were fractionated through 1.0% denaturing formaldehyde-aga-rose gels. RNA was transferred to a Hybond N� membrane (AmershamBiosciences) by capillary blotting with 10� SSC (1� SSC is 0.15 M NaClplus 0.015 M sodium citrate) (pH 7.0) for 16 h and fixed to the membraneby UV cross-linking at a wavelength of 302 nm for 3 min. Oligonucleo-tides pnpTL�11R (5=-GGATTAAGCAATGTAATATCCTTTCTC-3=)for pnp mRNA hybridization and 16SrRNAR (5=-GCAGGTTCCCCTACGGTTACCT-3=) for 16S rRNA hybridization, which served as a loadingcontrol, were 5= end labeled with [�-32P]ATP and T4 polynucleotide ki-nase (New England BioLabs). Hybridization was performed according tothe manufacturer’s instructions (Amersham Biosciences). Labeled RNAspecies were visualized with a phosphorimager and quantified usingImageJ software (36).

In vitro coupled transcription-translation assay. Plasmid pT7-pnpFl=-=lacZ contains a T7 promoter driving transcription of a pnp=-=lacZtranslational fusion (�156 to �182). This plasmid was designed such thattranscription initiated at the natural pnp transcription start site but withtwo additional G residues for initiation by T7 RNA polymerase (RNAP).Plasmid pT7-pnpPr=-=lacZ was constructed in a similar manner exceptthat transcription began at �81, thereby mimicking a pnp transcript thatwas 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 templatesfor coupled transcription-translation reactions using a PURExpress invitro protein synthesis kit according to the instructions of the manufac-turer (New England BioLabs). Reaction mixtures containing 20 nM plas-mid DNA template and various concentrations of purified His-taggedCsrA were incubated for 2 h at 37°C. -Galactosidase activity was deter-mined according to the manufacturer’s instructions.

RESULTSCsrA represses pnp expression. PNPase is a 3=-to-5= exoribonu-clease and a component of the E. coli degradosome (23, 24).PNPase participates in an autoregulatory mechanism that alsoinvolves RNase III. RNase III-mediated cleavage of a pnp leader

FIG 1 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 PNPasebecause 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 basepairing 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 represspnp translation (right). The highly conserved GGA motif of each CsrA binding site is highlighted. Positions of the Shine-Dalgarno (SD) sequence and translationinitiation codon (Met) are also shown. RNA structural predictions were carried out with MFOLD (44) using constraints based on RNA structure-mappingexperiments presented here.

FIG 2 CsrA represses pnp expression. Expression of chromosomally inte-grated wild-type (WT) and BS1 mutant pnp=-=lacZ fusions was examined inWT or csrA mutant strains. -Galactosidase activity (Miller units) standarddeviation is shown with solid lines, while growth (optical density at 600 nm[OD600]) is shown with dashed lines. Experiments were performed at leastthree times.

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RNA structure removes the top portion of the stem such that thetranscript containing the downstream pnp coding sequence re-mains base paired to the upstream (5=) cleavage fragment (Fig. 1).PNPase then degrades the 5= fragment from the newly generated3= end, resulting in a pnp transcript that is regulated by an un-known translational repression mechanism (25, 26). Our previ-ously published RNA-seq studies identified pnp as a CsrA target(19). Visual inspection of the untranslated leader region of pnpmRNA identified two putative CsrA binding sites, each containingthe highly conserved GGA motif. The downstream site (BS2)overlaps the pnp SD sequence, suggesting that CsrA might be ca-pable of repressing pnp translation. Prior to the action of RNase IIIand PNPase, the upstream site (BS1) would be sequestered in thelarge secondary structure, perhaps inhibiting CsrA binding (Fig.1). Thus, we tested a model in which CsrA participates in the pnpregulatory mechanism by repressing translation following the ac-tion of RNase III and PNPase. Translational repression could thenlead to accelerated decay of pnp mRNA.

We first examined expression of a pnp=-=lacZ translational fu-sion in wild-type and CsrA-deficient strains. Expression in thewild-type background was low throughout growth. In contrast,expression of the fusion in the csrA mutant background increaseddramatically in exponential phase and remained high in stationaryphase, indicating that csrA represses pnp expression throughout

growth, with maximal 20-fold repression occurring during sta-tionary phase (Fig. 2). We also examined expression of a fusion inwhich the GGA motif in BS1 was replaced with GAG. This mutantleader was designed to prevent CsrA binding to BS1 while main-taining base pairing of the long stem recognized by RNase III (Fig.1, left panel). Expression of the mutant fusion was �5-fold higherthan expression of the wild-type fusion, suggesting that CsrA isunable to fully repress pnp expression when CsrA is not bound toBS1. We did not test mutations in BS2 since mutations in this sitewould also disrupt the pnp SD sequence.

CsrA binds to fully processed pnp mRNA. To determinewhether processing of the pnp leader transcript by RNase III andPNPase affects CsrA binding, we performed quantitative gel mo-bility shift assays with two in vitro-generated transcripts, both ofwhich contained BS1 and BS2. One transcript contained the full-length pnp leader (pnpFl), while the 5= end of the other transcriptbegan at the 3= RNase III cleavage site (pnpPr), thereby mimickingthe transcript produced in vivo following RNase III and PNPaseprocessing (Fig. 3A). CsrA bound tightly to fully processed pnpPr

RNA with an apparent Kd of 20 nM. In contrast, only weak bind-ing to full-length pnpFl RNA was observed at the highest CsrAconcentration tested (Fig. 3B and E). The specificity of CsrA-pnpPr

RNA interaction was investigated by performing competition ex-periments with specific (pnpPr) and nonspecific (phoB) unlabeled

FIG 3 Gel mobility shift analysis of CsrA-pnp leader RNA interaction. (A) Two 5=-end-labeled RNAs were tested that mimic the unprocessed full-length pnpleader (pnpFl) and the fully processed leader that results following RNase III cleavage and exonucleolytic degradation by PNPase (pnpPr) (Fig. 1). (B) Gel shiftassay 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 ofbound (B) and free (F) RNA are also shown. (C) RNA competition experiment demonstrating binding specificity. Labeled pnpPr RNA was incubated with theindicated 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 atthe 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.

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RNA competitors. Unlabeled pnpPr was an effective competitor,whereas phoB was not, indicating that the CsrA-pnp RNA interac-tion 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 (BS2mutant) with an apparent Kd of 57 nM, while the affinity of CsrAfor the transcript containing BS2 only (BS1 mutant) was about2-fold lower. Binding was not observed to the transcript contain-ing mutations in both binding sites.

CsrA-pnpPr RNA footprint experiments were performed toverify CsrA binding to BS1 and BS2. Bound CsrA reduced RNaseT1-mediated cleavage of G residues at positions �73, �71, �70,�67, and �65 within BS1. Similarly, CsrA prevented cleavage ofpositions �10 and �9 within BS2 (Fig. 4). In addition to protec-

tion of BS1 and BS2, bound CsrA caused increased cleavage of G3within the UUG start codon and at positions G16 and G20 furtherdownstream, indicating that CsrA binding alters the RNA struc-ture in the translation initiation region. The finding that CsrAprotected BS1 at the lowest concentration of CsrA used in thisanalysis, while protection of BS2 required a higher CsrA concen-tration, is consistent with our gel shift analysis demonstrating thatCsrA has higher affinity for BS1 (Fig. 3D). In addition, our struc-ture mapping data in the control lane without CsrA (Fig. 4) areconsistent with the two CsrA binding sites being single stranded inthe fully processed RNA, as depicted in our RNA structural model(Fig. 1, right panel). Taken together, our binding studies indicatethat BS1 and BS2 constitute authentic CsrA binding sites. More-over, our results demonstrate that CsrA binds to fully processedpnp mRNA with high affinity and specificity but not to the unpro-cessed pnp leader transcript.

CsrA represses pnp translation. There are two mechanisms inwhich CsrA has been shown to repress gene expression posttran-scriptionally. First, bound CsrA can decrease the stability of pnpmRNA. Second, CsrA can repress translation by blocking ribo-some binding. These two possibilities are not mutually exclusive,as destabilization of mRNA can be an indirect consequence oftranslation inhibition. We first examined the stability of pnpmRNA in WT and CsrA-deficient strains by Northern blotting.The pnp half-life durations in the wild-type and csrA mutantstrains were 4.3 and 6.3 min, respectively (Fig. 5). This differenceis too small to account for the large difference in expression ob-served in the pnp=-=lacZ translational fusion studies (Fig. 2).

We next used the in vitro coupled transcription-translationPURExpress system to determine whether CsrA inhibits pnptranslation. Three different plasmids carrying pnp=-=lacZ transla-tional fusions, all of which were driven by identical T7 RNA poly-merase promoters, were used in this analysis. One plasmid gave

FIG 4 CsrA-pnp leader RNA footprint analysis. (A) Sequence of pnp leaderRNA. Positions of the downstream RNase III cleavage site, CsrA binding sites1 (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 theconcentration 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 withoutRNase treatment (C), are shown. Positions of BS1, BS2, the translation initia-tion codon (Met), and residues in which bound CsrA showed reduced (�) orincreased (�) cleavage are marked.

FIG 5 Northern blot analysis of pnp mRNA half-lives in wild-type (WT) andcsrA mutant strains. Cultures were grown at 37°C to the mid-exponentialphase prior to the addition of rifampin. After 1 min, samples were harvested atthe indicated times. mRNA half-lives (T1/2) standard deviations are shownat the bottom of the gel. The level of pnp mRNA was normalized to the 16SrRNA level in each lane. Experiments were performed twice, and a represen-tative gel is shown. Quantifications of these data are shown in the bottompanel.

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rise to full-length pnp leader RNA (pnpFl) identical to that used inour in vivo expression studies (Fig. 2), while a second was designedto generate a transcript mimicking the fully processed pnp tran-script (pnpPr). These two transcripts were essentially identical tothose used in our gel shift analysis (Fig. 3A and B). The thirdconstruct contained the BS1 GGA-to-GAG mutation in pnpPr.Expression from each construct was measured by determining-galactosidase activity. Addition of CsrA to the system caused aslight decrease in expression of the pnpFl construct; expression wasreduced only 3% and 19% at 2.5 and 10 M CsrA, respectively(Fig. 6). In contrast, expression of the pnpPr construct was reduced54% and 91% at the same two CsrA concentrations. Expression ofthe pnpPr construct containing the BS1 mutation exhibited anintermediate level of CsrA-mediated repression. We conclude thatbound CsrA represses translation of pnp but only after the RNAhas been fully processed by RNase III and PNPase. These data alsosuggest that the small effect of CsrA on pnp mRNA stability (Fig.5) was an indirect consequence of altered translational repressionor RNA secondary structure.

CsrA-mediated repression of pnp expression requires priorprocessing by RNase III and PNPase. Our data described aboveare consistent with a model in which CsrA represses translation ofpnp 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 bindingsites are sequestered in the RNA secondary structure. However, inthe unprocessed transcript, the pnp SD sequence would be seques-tered in an RNA structure, which would inhibit translation (Fig. 1,left panel). Following processing by these two nucleases, the pnpleader RNA refolds such that the SD sequence is single strandedand available for ribosome binding. However, this processed andrefolded transcript would then be subject to CsrA-mediated trans-lational repression since both of the CsrA binding sites are single

stranded. Since our model predicts that CsrA functions down-stream of RNase III and PNPase, we expected that disrupting rncor pnp would suppress the effect of the csrA mutation on pnpexpression. Thus, epistasis studies were carried out to test thismodel by examining expression of a pnp=-=lacZ translational fu-sion containing the entire leader region. Since processing of thepnp transcript is initiated by RNase III-mediated cleavage, we firstcompared 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 repressionof the fully processed transcript. We also found that expressionwas elevated in the rnc strain, indicating that sequestration of thepnp SD sequence in the unprocessed mRNA is less effective atrepressing translation than CsrA-mediated repression of pro-cessed transcripts (Fig. 7A). Notably, as predicted by our model,the large increase in expression of the csrA mutant strain was com-pletely suppressed in the RNase III-deficient background. How-ever, we cannot account for the unexpected result that expressionin the csrA rnc double-mutant strain was actually 2-fold lowerthan expression in the rnc strain.

We next compared expression levels in the WT, csrA, pnp, andcsrA pnp strains (Fig. 7B). Expression was elevated in the pnp mu-tant strain but to a much smaller extent than in the csrA strain.Importantly, as predicted by our model, the large increase of ex-pression in the csrA mutant strain was completely suppressed inthe PNPase-deficient background; expression levels in the pnp andpnp csrA strains were essentially identical. Taken together with theresults of our in vitro studies, we conclude that CsrA is effectiveonly at binding to and repressing translation of pnp transcriptsthat are fully processed by RNase III and PNPase.

DISCUSSION

PNPase is one of three 3= to 5= exoribonucleases responsible forthe degradation of bulk mRNA in E. coli (reviewed in reference37). The other two enzymes, RNase II and RNase R, degrade RNAby a hydrolytic mechanism with the release of nucleoside mono-phosphates, whereas PNPase degrades RNA through a phospho-rolytic mechanism, resulting in the release of nucleoside diphos-phates. PNPase is also capable of catalyzing the reverse reaction,thereby synthesizing RNA from nucleoside diphosphates. This ac-tivity is responsible for the synthesis of polynucleotide tails on the3= ends of transcripts in strains lacking PAP I, the enzyme nor-mally responsible for adding poly(A) tails; single-stranded 3= tailsprovide a toehold for 3= to 5= exonucleases to degrade structuredRNA (38). Although PNPase is capable of degrading RNA as atrimer of identical subunits, this enzyme also functions as acomponent of the degradosome. This multisubunit complexalso contains the endoribonuclease RNase E, which serves asthe scaffold for degradosome assembly, the DEAD box RNAhelicase RhlB, and the glycolytic enzyme enolase (23, 24). Atlow temperatures, an alternative degradosome forms in whichDeaD (CsdA) replaces RhlB (39). PNPase also exists as a com-plex containing the PNPase homotrimer and two subunits ofRhlB (40). The unwinding activity of a DEAD box RNA heli-case in each of these complexes probably enables PNPase todegrade structured RNA substrates (37).

pnp expression is repressed by a mechanism that was previ-ously shown to involve RNase III-mediated cleavage of a large

FIG 6 Effect of CsrA on pnp translation. Coupled transcription-translationreactions were performed with a PURExpress kit using WT pnpFl, WT pnpPr,and BS1 mutant pnpPr DNA templates containing pnp=-=lacZ translationalfusions. Purified CsrA protein was added prior to starting the reaction.-Galactosidase activity normalized to 0 M CsrA for each template isshown. Each experiment was performed at least twice, with representativeresults shown.

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secondary structure that forms in the untranslated leader of thepnp transcript, resulting in a 5= fragment that remains base pairedto the downstream sequence (Fig. 1). This 5= fragment contains a2-nucleotide (2-nt) 3= extension that serves as a substrate for exo-nucleolytic digestion by PNPase (25, 26). It was noted that, fol-lowing 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). Wefound that CsrA binds to two sites in the pnp transcript that is fullyprocessed by RNase III and PNPase (Fig. 3 and 4). The affinity ofCsrA was highest for BS1, which is 61 nt upstream of BS2, whichoverlaps the SD sequence. It was previously shown that a singleCsrA dimer is capable of bridging a high-affinity site to a lower-affinity site (6). Thus, it is apparent that dual-site bridging partic-ipates in the mechanism leading to dramatic repression of pnptranslation (Fig. 2 and 6). In the absence of processing by RNaseIII and PNPase, the critical GGA motif of both CsrA binding sitesremains sequestered in the RNA secondary structure (Fig. 1 and4). Since CsrA binding requires that the GGA motif is unpaired (3,5, 8), CsrA is unable to bind to unprocessed transcripts (Fig. 3 and6). Indeed, the large increase in pnp expression observed in thecsrA mutant is suppressed by mutations in either rnc or pnp, indi-cating that the ability of CsrA to repress pnp expression is elimi-nated in strains lacking RNase III or PNPase (Fig. 7). However, thefinding that pnp expression was higher in the rnc strain than in thepnp strain (Fig. 7) indicates that our model does not fully accountfor the effects of these two enzymes. This is not surprising, as onewould expect these globally acting nucleases to affect pnp expres-sion by a variety of direct and indirect mechanisms. For example,a recent study found that PNPase directly represses its translationin an RNase III-independent manner (42).

CsrA-mediated repression of pnp translation was observedthroughout growth, although the extent of the repression wasmost pronounced in stationary phase (Fig. 2 and 7). The increasein repression as cells exit exponential growth parallels the previ-ously observed activation of csrA transcription mediated by RpoS(�S), the general stress response sigma factor of RNA polymerase(13). Under our growth conditions, the concomitant increases inCsrA levels and CsrA-mediated translational repression resultedin similar levels of pnp expression throughout growth (Fig. 2 and7). This finding suggests that E. coli evolved an elaborate mecha-nism to maintain near-constant levels of PNPase in the cell. How-ever, as CsrA activity is regulated by its two sRNA antagonistsCsrB and CsrC (7, 14), alternative growth conditions or stressesleading to rapid changes in CsrB and CsrC levels could result influctuations in PNPase levels. For example, the Csr system recip-rocally regulates the stringent response (19), which is character-ized by a rapid downshift in ribosome biogenesis in response toamino acid starvation (reviewed in reference 43). Of particularinterest, DksA and ppGpp, the mediators of the stringent re-sponse, activate csrB and csrC transcription 10-fold, whereas CsrArepresses translation of relA, the gene encoding one of two ppGppsynthases. This complex circuitry suggests that CsrA-mediatedregulation of gene expression is relieved during the stringent re-sponse (19). Thus, amino acid starvation, and perhaps other stressconditions that increase levels of CsrB and CsrC, is predicted toresult in higher levels of PNPase.

ACKNOWLEDGMENTS

Hongmarn Park performed experiments, interpreted results, and wrotethe manuscript. Helen Yakhnin performed experiments and interpretedresults. Michael Connolly performed experiments and interpreted results.Tony Romeo designed the study, edited the manuscript, and securedfunding. Paul Babitzke designed and supervised the study, wrote the man-uscript, and secured funding.

FIG 7 Effects of CsrA, RNase III, and PNPase on pnp expression. -Galacto-sidase activity (Miller units) standard deviations of a chromosomally inte-grated pnp=-=lacZ translational fusion is indicated (solid lines). A representa-tive growth curve is shown for each strain (dashed lines). Each experiment wasperformed 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.

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We thank Louise McGibbon for critical reading of the manuscript andSidney R. Kushner for providing strain SK10019 containing the pnp�683::(Str Spr) allele.

This work was supported by National Institutes of Health grantGM059969 to Tony Romeo and Paul Babitzke.

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