Index INTRODUCTION MATERIALS AND METHODS E. coli strains, phages, plasmids and primers Bacterial growth and Northern and Western analyses Ligation-mediated RT-PCR MALDI-TOF/MS In vitro transcription RESULTS Cleavage of arfA mRNA by RNase III Tagging and proteolysis of ArfA in wild type cells Instability of full-length ArfA Expression of chromosomally encoded ArfA DISCUSSION References

INTRODUCTION

In the process of translation termination, peptidyl-tRNA is hydrolyzed by the action of release factors (RF) which recognize stop codons (for review, see Youngman et al., 2008). When ribosome translates mRNA without an in-frame stop codon (non-stop mRNA), canonical translation termination cannot take place and ribosome stalls at the 3' end of non-stop mRNA. In bacteria, accumulation of stalled ribosomes is deleterious and thus such ribosomes are rescued by SsrA-mediated trans-translation (Keiler et al., 1996) (for reviews, see Moore and Sauer, 2007; Keiler, 2008; Hayes and Keiler, 2010). SsrA is a small RNA and also called tmRNA because it has both tRNA- and mRNA-like domains. In trans-translation, SsrA charged with alanine at its CCA end binds to the unoccupied A-site of the ribosome and receives a nascent polypeptide by the peptidyl-transfer reaction as regular tRNAs do in translation. Then ribosome switches its template from non-stop mRNA to the mRNA-like domain of SsrA and translation proceeds to and terminates at the stop codon on SsrA. As a result, stalled ribosomes are released from non-stop mRNA. During this process, translated polypeptides receive the SsrA-coded “degradation tag” (also called “SsrA-tag”) at their C-termini and will be degraded by cellular proteases. This enables cells to remove potentially harmful truncated polypeptides coded by non-stop mRNAs. This feature is called a protein quality control function of trans-translation. Also, removal of ribosomes enhances degradation of non-stop mRNAs (Yamamoto et al., 2003). Thus, trans-translation deals with the problems arising from non-stop mRNAs.

SsrA is widely distributed among eubacteria (Moore and Sauer, 2007; Withey and Friedman, 2003), suggesting that trans-translation plays an important role in bacterial cells. In fact, SsrA is required for the growth of several bacterial species including Neisseria gonorrhoeae (Huang et al., 2000), Haemophilus influenzae (Akerley et al., 2002) and Mycoplasma genitalium (Glass et al., 2006). Curiously, however, SsrA is known to be dispensable for the growth in many other bacterial species including Escherichia coli (Komine et al., 1994).

Although SsrA is dispensable for the growth of E. coli, SsrA-deficient E. coli cells show several phenotypes including growth defect at extremely high temperature (Komine et al., 1994), lowered level in phage propagation (Withey and Friedman, 1999; Ranquet et al., 2001), delayed induction of the lac operon (Abo et al., 2000), increased sensitivity to several antibiotics (Abo et al., 2002; Luidalepp et al., 2005) and growth inhibition under amino-acid starvation (Li et al., 2008). It is noteworthy that all of these phenotypes could be recovered by SsrA^DD, an SsrA variant which adds a protease-resistant sequence (AANDENYALDD) instead of the degradation tag (AANDENYALAA) during trans-translation (Keiler et al., 1996). This suggests that the ribosome rescue function is more critical than the protein quality control function at least in E. coli.

We have recently reported that E. coli ArfA also rescues stalled ribosome in the manner distinct from trans-translation (Chadani et al., 2010). ArfA-mediated ribosome rescue is not accompanied with degradation of nascent polypeptide. Although neither ArfA nor SsrA is essential for the cell viability, combination of arfA and ssrA mutations is synthetically lethal. This suggests that ribosome rescue is quite important for E. coli cells. In the same report, we mentioned that N-terminally His₆-tagged full length ArfA could not be efficiently expressed, whereas a His₆-ArfA protein lacking C-terminal 12 amino acids was expressed well. Furthermore, we showed that ArfA-mediated ribosome rescue was predominant only when SsrA was absent. In this study, we found that arfA mRNA was expressed as a truncated form due to the premature transcription termination and specific cleavage by RNase III within its open reading frame (ORF). arfA non-stop mRNA was targeted by trans-translation and the synthesized truncated ArfA protein was degraded. On the other hand, full length ArfA expressed from mRNA, which escaped from premature transcription termination and RNase III cleavage, was shown to be unstable. These data suggest that ArfA expression is tightly repressed when capacity of trans-translation is high enough, which conforms to a hypothesis that ArfA-mediated ribosome rescue is the backup system of trans-translation.

MATERIALS AND METHODS

E. coli strains, phages, plasmids and primers

E. coli strains, plasmids, and primers used in this study are listed in Table 1, Table 2, and Table 3, respectively. Phage P1-mediated transduction was used to introduce the rnc mutation from ST201 to appropriate strains. pQE80L (Qiagen) was used to construct plasmids carrying inducible arfA. The pCH200 variants (pCH200dC6, dC17, dC22, dC25 and dC32) were constructed by cloning BamHI- and PstI-digested PCR fragments amplified using primer pairs LF02_BamHI and yhdL-dC6PstIRV, LF02_BamHI and yhdL-dC17PstIRV, LF02_BamHI and yhdL-dC22PstIRV, LF02_BamHI and yhdL-dC25PstIRV and LF02_BamHI and yhdL-dC32PstIRV, respectively, and pCH200 as a template into the BamHI and PstI region of pQE80L.

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Table 1 Strains used

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Table 2 Plasmids used

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Table 3 Primers used

A 177-bp DNA fragment PCR amplified from pCH200 using a primer pair LF02_BamHI and stem1_rv and a 155-bp DNA fragment PCR amplified from the same plasmid using a primer pair LR01_PstI and stem1_fw were mixed and used as a partially overlapping template for PCR reaction using a primer pair LF02_BamHI and LR01_PstI. The resulting 302-bp DNA fragment was digested with BamHI and PstI and cloned into the corresponding site of pQE80L to obtain pCH200stem1.

A 227-bp DNA fragment PCR amplified from pCH200stem1 using a primer pair LF02_BamHI and stemDD_rv and a 93-bp DNA fragment PCR amplified from the same plasmid using a primer pair LR01_PstI and stemDD_fw were mixed and used as a partially overlapping template for a PCR reaction using a primer pair LF02_BamHI and LR01_PstI. The resulting 302-bp DNA fragment was digested with BamHI and PstI and cloned into the corresponding site of pQE80L to obtain pCH200stemDD. pSTV29-arfA-EE was constructed by cloning the EcoRI- and EcoT22I-digested PCR fragment amplified using a primer pair LF05_EcoRI and M13RV and pL-4 as a template into the EcoRI-PstI region of pSTV29.

pSPT-arfA was constructed by cloning the BamHI- and PstI-digested PCR fragment amplified using LF02_BamHI and yhdL_dC12PstI RV as primers and pCH200 as a template into the BamHI-PstI region of pSPT18.

Bacterial growth and Northern and Western analyses

E. coli cells were grown in LB broth. If required, appropriate antibiotics were added to the culture medium. Bacterial growth was monitored by measuring OD₆₆₀.

For Northern blotting analysis, total RNA was prepared using TriPure Isolation Reagent (Roche) according supplier’s instruction. One μg of RNA was separated by 1.5% denaturing agarose electrophoresis and subsequently blotted to a nylon membrane. Digoxigenin (DIG)-labeled DNA probe hybridizing to the arfA gene was prepared with DIG DNA labeling kit (Roche) by PCR using a primer pair LF02_BamHI and LR01_PstI and pCH200 as a template. DIG-labeled RNA probe hybridizing to arfA mRNA or 5S rRNA was prepared with DIG RNA labeling kit (Roche) by transcribing pSPT-arfA digested with EcoRI or pRT18 digested with EcoRI, respectively.

For Western blotting analysis, plasmid-borne and endogenous ArfA proteins were detected by anti-His₆ antibody (Roche) and anti-ArfA antibody, respectively. Briefly, cells cultured in LB medium were harvested at late-log phase and washed with ice-cold STE (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA). Then cells were suspended in disruption buffer (50 mM NaH₂PO₄ pH 7.0, 150 mM NaCl, 8 M urea) and disrupted by sonication. After centrifugation, proteins in supernatant were separated by 20% SDS-PAGE and subsequently transferred to a nitrocellulose membrane. The membrane was blocked by 3% skim milk in TBS-Tween (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) at room temperature for 1 hr. Then the membrane was incubated with TBS-Tween containing antibody (1/3000 diluted) at room temperature for 1 hr. Horseradish peroxidase-conjugated Rabbit IgG (abcam) secondary antibody and ECL plus (GE Healthcare) were used for chemiluminescence detection.

Ligation-mediated RT-PCR

Ligation-mediated RT-PCR was performed as described by Yonesaki (2002) with some modifications. RNA prepared from exponentially growing TA331 (ΔssrA), ST201 (Δrnc) or CH101 (ΔarfA) was dephosphorylated using alkaline phosphatase (calf intestine), phosphorylated by T4 nucleotide kinase and subjected to T4 RNA Ligase reaction. Then the sample was subjected to reverse transcription (RT) reaction using LR02_EcoRI as a primer DNA. The RT product was then used as a template in PCR using a primer pair LR02_EcoRI and LF03_PstI. The amplified DNA fragment was digested with EcoRI and PstI and cloned into corresponding site of pSTV28, and the inserted DNA was sequenced.

MALDI-TOF/MS

Cells harboring pCH200 (ArfA-FL) was grown in LB. At early-log phase, isopropyl-thiogalactoside (IPTG) was added to the final concentration of 500 μM and cells were incubated for 2 hr to induce protein expression. Cells were harvested, washed with ice-cold STE and then suspended in buffer A (25 mM HEPES-KOH pH 7.4, 1 M NaCl, 10% glycerol, 2 M urea, 10 mM imidazole, 3 mM 2-mercaptoethanol, 0.2 mM phenylmethanesulfonyl fluoride; 1 ml for 100 mg cell). Lysozyme was added to the final concentration of 250 μg/ml, and samples were incubated for 30 min on ice. Then the cells were disrupted by sonication. Debris was sedimented by centrifugation (15 krpm, 10 min, 4°C). Supernatant was subjected to TALON Metal Affinity resin (Clontech) chromatography to purify N-terminally His₆-tagged ArfA. Resin was washed six times with buffer B (25 mM HEPES-KOH, 1 M NaCl, 25 mM imidazole). His₆-ArfA was eluted by 0.3% trifluoroacetic acid (TFA) and desalted by C-tip (AMR) according to the supplier’s instruction. Then, 1 μl of eluate (in 90% acetonitrile, 0.1% TFA) and 10 μl of 3,5-dimethoxy-4-hydroxycinnamic acid (0.3 mg/ml in ethanol/acetone 2:1, 0.1% TFA) were mixed and dropped onto an AnchorChip^TM Targets plate (Bruker Daltonics) and co-crystallized at room temperature. Then samples were subjected to MALDI-TOF/MS using Autoflex (Bruker Daltonics) in linear mode, and obtained data were analyzed by flexAnalysis (Bruker Daltonics).

In vitro transcription

In vitro RNA synthesis was performed using σ⁷⁰-saturated E. coli RNA polymerase holoenzyme (Epicenter Biotechnologies, WI) according to the method described previously (Yamamoto and Kutsukake, 2006). The reaction mixture contained 40 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM DTT, 10 mM MgCl₂, 50 U of RNase inhibitor (Promega), 100 μM each ATP, GTP and CTP and 50 μM UTP with 4 × 10⁵ Bq [α-³²P] UTP (Institute of Isotopes, Budapest, Hungary), 1 μg of template DNA and 1 U of RNA polymerase. HindIII- or HindIII- and EcoRV-digested pSTV29-arfA-EE was used as a DNA template. Mixtures without substrates were prepared and incubated at 37°C for 20 min. The reaction was initiated by addition of pre-warmed substrate mixture. After incubated at 37°C for 20 min, the reaction was terminated by adding a stop solution (0.6 M sodium acetate [pH 5.5], 20 mM EDTA, 200 μg tRNA/ml). The transcripts were precipitated with ethanol and separated in a 6% polyacrylamide gel containing 6 M urea.

An RNA ladder was synthesized using RNA Century-Plus Marker Templates (Ambion) as a template according to manufacturer’s instruction. The labeled transcripts were visualized by autoradiography.

RESULTS

Cleavage of arfA mRNA by RNase III

We constructed plasmids encoding full-length His₆-ArfA (ArfA-FL) and its truncated variants that lack C-terminal 6, 12, 17, 22, 25 and 32 amino acids (ArfA-dC6, dC12, dC17, dC22, dC25 and dC32, respectively) (Fig. 1A). When these proteins were expressed in E. coli cells, amounts of ArfA-FL, ArfA-dC6 and ArfA-dC32 were very low, whereas those of the other constructs were high (Fig. 1B, top). Amounts of their mRNAs showed a similar pattern (Fig. 1B, middle). These observations suggested that arfA has a regulatory element in its promoter-distal region which is present in genes for ArfA-FL and ArfA-dC6 but absent from genes for ArfA-dC12 to dC32. Analysis by mfold (Zuker, 2003) revealed that arfA mRNA has a stable stem-loop structure just upstream of the stop codon of arfA ORF (Fig. 1C). This structure is present in genes for ArfA-FL and ArfA-dC6 and absent from genes for ArfA-dC12 to dC32 mRNAs, suggesting a hypothesis that this stem-loop structure lowers the arfA expression. To test this, we introduced base-substitution mutations without changing amino acid sequence into the stem region (Fig. 1C). The mutant (ArfA-stem1) gene produced significant amounts of arfA mRNA and ArfA protein (Fig. 1B), supporting the hypothesis mentioned above. Low level expression of ArfA-dC32 may be due to its instability caused by large truncation.

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Fig. 1 Regulatory element in 3' region of arfA. (A) Schematic drawing of N-terminally His6-tagged ArfA protein and its derivatives used in this study. C-terminal hydrophobic region of ArfA is shown by filled box. Each ArfA mutant has the part of ArfA indicated by horizontal line and has His6-tag fused to the N-terminus. Horizontal convergent arrows indicate the inverted repeat which can form stem-loop structure. Dotted arrows above the lines for stem1 and stemDD indicate that the inverted repeat is disrupted by base substitution. ArfA-stemDD has two successive aspartates at its C-terminus. (B) Expression of ArfA variants shown in panel A. ArfA was induced by 500 μM IPTG and detected by Western blotting using anti-His6 antibody. arfA mRNA was detected by Northern blotting using arfA probe. Northern blotting using 5S rRNA probe was also shown as an internal control. (C) Stem-loop structure formed just before stop codon of arfA ORF. A representative secondary structure model of arfA mRNA predicted by mfold program (Zuker, 2003) is shown. Positions of stop codons introduced to construct C-terminally truncated ArfA variants and base substitution mutations in “stem” variants were indicated. (D) Expression of arfA mRNA in the absence of cellular nuclease. Each strain with the mutation indicated harboring pCH200 (ArfA-FL) was cultured in LB medium. Expression of ArfA was induced by 500 μM IPTG and arfA mRNA was analyzed by Northern blotting using arfA probe. Strains used: rna (RNase I), JW0603; rnb (RNase II), JW1279; rnc (RNase III), ST201; rnd (RNase D), JW1793; ams-1 (RNase E), GW20; rng (RNase G), GW11; rnr (RNase R), JW5741.

Several RNases are known to degrade double stranded RNAs. To see if the stem-loop structure of arfA mRNA is cleaved by a specific RNase, we analyzed ArfA-FL mRNA in various E. coli mutants lacking each one of RNases. As shown in Fig. 1D, ArfA-FL mRNA was detectable only in the rnc background, indicating that arfA mRNA is cleaved by RNase III, the rnc gene product. This conclusion is supported by the structural feature of the stem-loop region (paired region and bulge), which was reported to be a typical structure of RNase III substrate (Régnier and Grunberg-Manago, 1990).

Tagging and proteolysis of ArfA in wild type cells

Cleavage of arfA mRNA by RNase III at the stem-loop structure may produce arfA non-stop mRNA that will be targeted by trans-translation. To evaluate this, we compared the ArfA-FL expression among wild type, ΔssrA and ssrA^DD backgrounds (Fig. 2A). ArfA-FL mRNA and protein were detected in much higher amounts in the ΔssrA background than in the wild type background. This is consistent with the previous observation that both non-stop mRNA and its translation product are stable in the ΔssrA background (Chadani et al., 2010; also see Keiler, 2008 for review). In the ssrA^DD background, where trans-translation products will receive a proteolysis-refractory “DD-tag” and escapes from degradation, DD-tagged ArfA was detected (Fig. 2A, lanes 3 and 3'). Non-tagged ArfA was also detected in the ssrA^DD background. Similar result was obtained previously (Chadani et al., 2010). These results can be explained by lowered activity of SsrA^DD as reported by Mehta et al. (2006). Existence of substantial amount of arfA mRNA in the ssrA^DD background is consistent with this. It is also possible that DD-tagged truncated ArfA rescued the ribosomes stalled at the 3' end of arfA non-stop mRNA. Taken together, it is strongly suggested that arfA mRNA is targeted by trans-translation.

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Fig. 2 arfA mRNA is targeted by trans-translation in vivo. (A) W3110, TA331 (ΔssrA) and TA371 (ssrADD) harboring pCH200 (ArfA-FL) were grown in LB medium and expression of ArfA-FL was induced by 500 μM IPTG. ArfA was detected by Western blotting using anti-His6 antibody, and arfA mRNA was detected by Northern blotting using arfA probe. ArfA purified from TA371 (ssrADD) was also analyzed by Western blotting using anti-DD antibody (Abo et al., 2000). (B) MALDI-TOF Mass-spectrometry analysis of ArfA purified from TA331 (ΔssrA) and TA371 (ssrADD). Co2+-affinity purified ArfA was directly subjected to MALDI-TOF/MS. Mass values correspond to ArfA products are indicated. (C) Nucleotide sequence of the E. coli arfA locus. Uppercase letters in nucleotide sequence indicate arfA ORF. Amino acid sequence of ArfA is also shown. Intermittent horizontal arrows indicate the position of inverted repeat. Upward arrows indicate the nucleotide substitutions introduced to disrupt the inverted repeat in stem variants. C-terminal hydrophobic region is indicated by bold face letters with underline. Predicted –35 and –10 sequences are boxed. Downward filled and upward open triangles indicate the 5' and 3' ends of arfA mRNA mapped by ligation-mediated RT-PCR analysis, respectively (Table 4). C-termini of ArfA mapped by MALDI-TOF/MS analysis are highlighted by black (53A and 54S that gave strong signals) and gray (51W, 52E and 55G that gave minor signals) boxes.

The cleavage position of chromosomally encoded arfA mRNA produced in ΔssrA cells was determined by ligation-mediated RT-PCR technique. Thus mapped 5' and 3' ends of arfA mRNA are summarized in Table 4 and Fig. 2C. The 3' end was mapped within the stem region (Fig. 2C, nucleotides 159–164, where nucleotide number starts at the first A of the arfA ORF). This position matches the previously reported motif for RNase III cleavage site (Régnier and Grunberg-Manago, 1990). In rnc cells, the 3' ends were mapped at different positions (nucleotides 201–204). The former positions may reflect the specific cleavage site of RNase III, but the latter is independent of RNase III. This suggests that transcription of arfA mRNA is prematurely terminated even in the absence of RNase III. As shown in Fig. 1C, long stem-loop structure followed by U-stretch can be formed in this region of arfA mRNA. We anticipate that downstream transcription termination occurs in this region. Considering that the latter positions were only detected in rnc mutant cells and that the prematurely terminated transcripts still can form stem-loop structure, most arfA mRNA may be cleaved by RNase III irrespectively of premature transcription termination. Thus, arfA mRNA is strictly programmed to be non-stop.

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Table 4 Summary of 5'- and 3'-end mapping of arfA mRNA

His₆-ArfA proteins produced from pCH200 (ArfA-FL) in ΔssrA and ssrA^DD cells were purified using Ni-chelating resin and subjected to MALDI-TOF/MS analysis (Fig. 2B). Based on the molecular mass of the samples, C-terminal end of ArfA proteins produced in the ΔssrA background was mapped to W^51st to G^55th, where major signals were obtained for A^53rd and S^54th. This was in good agreement with the cleavage positions of arfA mRNA mapped above. Molecular mass of His₆-ArfA proteins produced in the ssrA^DD background gave two major peaks (Fig. 2B). One of the major peaks showed the same mass as that obtained for His₆-ArfA proteins produced in the ΔssrA background, corresponding to non-tagged His₆-ArfA proteins as shown in Fig. 2A, lane 3. The other showed the mass value that is in good agreement with the value if DD-tag was attached to the C-terminal end of the non-tagged ArfA proteins mentioned above. Truncated ArfAs whose C-terminal end is around L^68th or S^69th, corresponding to nucleotides 201-204, were not detected. This is consistent with the idea that prematurely terminated arfA non-stop mRNA (with its 3' end located at nucleotides 201–204) is further subjected to RNase III cleavage.

Instability of full-length ArfA

Amount of ArfA proteins produced from the stem1-mutant arfA gene (ArfA-stem1) was significantly lower than that of the truncated ArfA proteins (Fig. 1B, lane 8). The ArfA protein has an extremely hydrophobic C-terminal tail (Fig. 2C) which might destabilize this protein (Parsell et al., 1990). To test if the C-terminal tail destabilizes the ArfA protein, we analyzed the expression of ArfA-stemDD which has two successive aspartates instead of alanine and cysteine at the C-terminus of ArfA-stem1. As expected, ArfA-stemDD produced in wild type cell was more abundant than ArfA-stem1 (Fig. 1B, lane 9). Half-lives for ArfA-stem1 and ArfA-stemDD were 1.58 min and 30.89 min, respectively (Fig. 3), indicating that full-length ArfA is unstable. We hypothesize that ArfA protein produced from arfA mRNA which escapes from both premature transcription termination and cleavage by RNase III is readily degraded. According to this hypothesis, expression of ArfA is tightly repressed when trans-translation sufficiently occurs and truncated forms of ArfA are produced when trans-translation is insufficient. Such truncated ArfA proteins lack C-terminal 17 to 22 amino acids as shown in Fig. 2. Ribosome-rescuing activity of ArfA-dC12 has been reported previously (Chadani et al., 2010) and ArfA-dC25 also has an equivalent activity (Fig. 4A), suggesting that truncated ArfA thus produced can act as a ribosome rescue factor.

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Fig. 3 Stability of ArfA-stem1 and ArfA-stemDD proteins. W3110 harboring pCH200stem1 or pCH200stemDD was grown in LB medium until mid-log phase, and expression was induced by 250 μM IPTG for 30 min. Then translation was inhibited by 200 μg/ml spectinomycin at time 0. Cells were sampled at each time point indicated and treated with 10% ice-cold trichloroacetic acid. Then protein sample was washed, resolved in SDS-urea sample buffer (50 mM Tris-HCl pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 0.01% bromophenol blue, 10% glycerol, 4 M urea) and separeted by SDS-PAGE. ArfA-stem1 and ArfA-stemDD proteins were detected by Western blotting using anti-His6 antibody. Western blotting using anti-L2 (RplB) was also shown as internal control. Band intensity was evaluated using Multi Gauge ver. 3.0 (FUJIFILM), plotted as shown below the gel, and the half-life of each ArfA variant was calculated. Average of three independent experiments are calculated as a half-life value and most representative profiles are shown.

Overexpression of ArfA in wild type cells had an inhibitory effect on growth (Fig. 4B). This suggests that ArfA-mediated ribosome rescue is not a preferable means for the E. coli cells. Cells may maintain the amount of ArfA as low as possible, especially in the presence of trans-translation system.

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Fig. 4 Biological function of ArfA variants. (A) Complementation of synthetic lethal phenotype of ssrA arfA double mutant by plasmid-borne His6-ArfA. CH111/pBAD33-ssrA harboring pQE80L (vector), pCH200 (ArfA-FL), pCH200dC6 (ArfA-dC6), pCH201 (ArfA-dC12), pCH200dC17 (ArfA-dC17), pCH200dC22 (ArfA-dC22), pCH200dC25 (ArfA-dC25) or pCH200dC32 (ArfA-dC32) was streaked onto LB agar plates containing arabinose (left) or glucose and 100 μM IPTG (right) and incubated for 20 hr at 37°C. (B) Inhibitory effect of overproduction of ArfA on the growth of E. coli cells. W3110 harboring pQE80L, pCH201 (ArfA-dC12) or pCH221 (ArfA-A18T-dC12: loss-of-function mutant) was cultured in LB medium and expression of ArfA was induced by 1 mM IPTG at early-log phase. Growth of each strain was monitored by measuring OD660.

The data obtained above suggest that ArfA is expressed only when the activity of trans-translation is limited. This predicts that ArfA is expressed when translation is somehow hampered. To see the effect of translation inhibition on ArfA expression, we treated growing cells with antibiotics and monitored the expression of His₆-ArfA proteins produced from pCH200 (ArfA-FL). As shown in Fig. 5, His₆-ArfA protein, presumably in the truncated form, was produced in the presence of sub-lethal concentration of chloramphenicol in wild type cells. In the ΔssrA background, ArfA was detected irrespective of chloramphenicol treatment.

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Fig. 5 Effect of chloramphenicol on ArfA expression. W3110 or TA331 harboring pCH200 (ArfA-FL) was cultured in LB medium, and expression of ArfA was induced by 250 μM IPTG at mid-log phase. Simultaneously, chloramphenicol was added at final concentrations of 0, 1.25, 2.5, 5 and 10 μg/ml. After incubating another 1 hr, ArfA in each culture was detected by Western blotting using anti-His6 antibody.

Expression of chromosomally encoded ArfA

The data so far presented were obtained using plasmid-encoded N-terminally His₆-tagged proteins. To evaluate our model, we analyzed the expression of the chromosomally encoded ArfA protein in various genetic backgrounds using anti-ArfA antibody. As shown in Fig. 6A, ArfA was detected in ΔssrA but not in wild type background (lanes 1 and 2). This indicates that the chromosomal arfA gene is also subjected to regulation by trans-translation. We assume the ArfA detected here was C-terminally truncated one. Chromosomally encoded arfA mRNA was much abundant in Δrnc ΔssrA strain than in Δrnc strain (compare lanes 4 and 5 in Fig. 6A), maybe because arfA non-stop mRNA is protected by ribosome stalled at its 3' end from degradation in the absence of SsrA. Consistently, chromosomally encoded ArfA was detected in Δrnc ΔssrA but not in Δrnc background. So we conclude that, even in the absence of RNase III, arfA mRNA is non-stop and targeted by trans-translation.

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Fig. 6 Expression of arfA. (A) Expression of chromosomally encoded arfA. W3110, TA331 (ΔssrA), CH101 (ΔarfA), ST201 (Δrnc) and CH371 (Δrnc ΔssrA) were cultured in LB medium and cells were harvested at late-log phase. Endogenous ArfA was detected by Western blotting using anti-ArfA antibody. arfA mRNA was detected by Northern blotting using DIG-labeled RNA probe for arfA. Northern blotting using 5S rRNA probe was also shown as internal control. (B) In vitro transcription of arfA. pSTV29-arfA-EE (its structure is shown below the gel) was transcribed by σ70-saturated E. coli RNA polymerase in the presence of [α-32P] UTP. Transcripts were separated by 6% polyacrylamide gel containing 6 M urea and detected by autoradiography. The bands for arfA mRNA and RNA I are indicated. The size of RNA ladder (lane M) is also shown.

Based on the position of 5' end of arfA mRNA revealed in RT-PCR experiment (Table 4), σ⁷⁰-dependent promoter could be predicted (Fig. 2C). In vitro transcription analysis using cloned arfA with its own 5'-UTR confirmed that it is transcribed by σ⁷⁰ RNA polymerase (Fig. 6B). The 3' end of in vitro transcript deduced from its length was in good agreement with the mapped position for 3' end of arfA mRNA in the absence of RNase III (Fig. 2C).

DISCUSSION

In this report, we showed that the expression of E. coli ArfA is regulated by the mechanism which includes RNase III-mediated non-stop mRNA production, premature transcription termination, trans-translation and subsequent protein degradation and intrinsic instability of full-length ArfA protein. All of these seem to act together to ensure that ArfA is produced only when trans-translation is somehow insufficient. This suggests that trans-translation is more preferable than ArfA-mediated ribosome rescue.

From the data obtained in this study, we illustrate a model that the arfA expression system monitors the activity of trans-translation and ArfA is expressed when the activity of trans-translation is lowered. In this model, trans-translation-mediated proteolysis plays a crucial role in arfA expression regulation by degrading truncated ArfA protein (Fig. 7). arfA is expressed as non-stop mRNA because of premature transcription termination and specific cleavage by RNase III. Ribosome stalls at the 3' end of arfA non-stop mRNA thus produced. In the regular condition, this ribosome is rescued by trans-translation and most of truncated ArfA will be degraded. If trans-translation cannot sufficiently occur, stalled ribosome would remain unreleased. This allows spontaneous drop-off of truncated ArfA from stalled ribosome. ArfB (YaeJ)-mediated ribosome rescue (Chadani et al., 2011; Handa et al., 2011) may also occur. In this way, insufficient trans-translation allows truncated but functional ArfA to be produced. This model can explain why SsrA-mediated ribosome rescue predominates ArfA-mediated one in wild type cells. If trans-translation sufficiently occurs, ArfA is not produced and stalled ribosomes are rescued by trans-translation. If ribosome stalls frequently enough to be beyond the capacity of trans-translation, truncated but functional ArfA will be produced and ArfA-mediated ribosome rescue takes place as a backup system for trans-translation.

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Fig. 7 Model for trans-translation-mediated regulation of ArfA expression. arfA is transcribed by σ70-RNA polymerase. Some of transcripts are terminated prematurely and the others contain a stop codon. Prematurely terminated transcript is non-stop. RNase III recognizes and cleaves the stem-loop structure of both transcripts, resulting in arfA non-stop mRNA production. Ribosome translating arfA non-stop mRNA stalls at its 3’ terminus and is rescued by trans-translation. As a result, SsrA-tagged truncated ArfA will be degraded. Full-length ArfA produced from arfA mRNA containing the stop codon is unstable and degraded immediately. Expression of ArfA is thus tightly repressed. When trans-translation is insufficient, however, stalled ribosome will be rescued by ArfB (YaeJ) or ArfA itself, producing truncated but functional ArfA which rescues stalled ribosomes.

Our model predicts that production of ArfA occurs only slightly in regular condition. Most ArfA mRNA is cleaved by RNase III or terminated prematurely at the stem structure. Full-length ArfA produced from arfA mRNA escaped from both RNase III cleavage and premature transcriptional termination is unstable and degraded. There should be the factor which rescues ribosome stalled at the 3' end of arfA non-stop mRNA. One possible candidate is ArfB (YaeJ), which rescues stalled ribosomes in the manner distinct from that of ArfA-mediated ribosome rescue or trans-translation (Chadani et al., 2011; Handa et al., 2011). Of course ArfA itself, SsrA-tagged or not, can lead its own expression once it is expressed.

Unlike the laboratory conditions, natural environment must be much more stressful to the bacterial cells. The stress includes high and low temperature, phages and other competitive organisms, host immune system and harmful compounds such as antibiotics, toxic chemicals and heavy metals. Existence of more than one ribosome rescue system suggests not only the importance of the maintenance of sound translation system but also high probability of ribosome stalling maybe caused by various environmental stresses. E. coli preferentially employs trans-translation system to rescue stalled ribosomes. This may be partly because of the growth inhibitory effect of ArfA. However, the fact that E. coli cells lacking SsrA can grow normally in regular laboratory conditions indicates that chromosomally encoded ArfA does not inhibit the cell growth. The amount of ArfA in the ΔssrA cells may be high enough for ribosome rescue but not so high as it shows inhibitory effect on the cell growth. The preference of trans-translation can be also explained by the difference in the output of ribosome rescue between trans-translation and ArfA-mediated system. Although both ArfA system and trans-translation rescue stalled ribosomes, the fate of nascent polypeptides differs: ArfA-mediated ribosome rescue leaves nascent polypeptides whereas trans-translation leads them to degradation. Shortage of amino acids or tRNA causes ribosome stall during translation (Garza-Sánchez et al., 2008; Li et al., 2008). Ribosome stalling induces mRNA breakage and produces the target of ribosome rescue (Sunohara et al., 2004). In such a situation, trans-translation is clearly preferable for the cells than ArfA-mediated ribosome rescue because degradation of nascent polypeptides provides amino acids which can be utilized in another round of translation. Considering this, tight regulation of ArfA expression in the presence of active trans-translation makes sense. Several phenotypes have been reported for SsrA-deficient E. coli cells as described in the “Introduction” section. We assume some of them are due to the lack of degradation of truncated proteins encoded by non-stop mRNAs. This also explains the preference of trans-translation.

There have been several reported examples in which stalled ribosome plays a role in gene regulation. Regulation of SecA expression by translational arrest within the preceding secM ORF (Nakatogawa and Ito, 2002) and TnaA production by the preceding tnaC ORF (Gong and Yanofsky, 2002) would be the best-studied examples. SecM-mediated ribosome arrest monitors the activity of SecA, and its shortage induces ribosome arrest and conformational change of mRNA which arrows expression of SecA. tnaC-mediated ribosome arrest adjusts the cellular concentration of tryptophan by controlling the expression of tryptophanase, TnaA. In both cases, ribosome arrest induces a conformational change of mRNA or physical hindrance of the regulatory element on mRNA and regulates the expression of downstream gene. In the case of ArfA, however, stalled ribosome does not seem to induce conformational change in arfA mRNA but is just targeted by trans-translation. Only when trans-translation falls insufficient in the cell, ArfA is expressed to rescue ribosome.

How this regulatory system has evolved? There is a clue to this question. Kobayashi et al. (2008) reported that the KinA protein was degraded in an SsrA-dependent manner in Bacillus subtilis when a base-substitution mutation in kinA ORF eliminated the stop codon. Although degradation of the KinA protein is just a result of mutation, it is intriguing to speculate that regulatory system for ArfA expression has its origin in such a mutation-promoted trans-translation. Being a back-up system for trans-translation, fine regulation system including programmed non-stop mRNA production must have been favorable to E. coli cells.

In the course of preparation of this manuscript, Garza-Sánchez et al. (2011) have published the similar results as we reported here. They also found that arfA is regulated by trans-translation and proposed a similar model for ArfA regulation. Our model includes intrinsic instability of full-length ArfA caused by its C-terminal hydrophobic region. This ensures very low amount of ArfA in the situation where ArfA is not needed. Also, we showed that the chromosomal arfA gene is regulated as predicted by the model. We believe our report presented here is still important to better understand the biological significance of ribosome rescue and the strategy that bacteria employ to deal with environmental stresses.

We thank Dr. Masaaki Wachi and NBRP: E. coli (NIG, Japan) for providing E. coli GW strains and JW strains, respectively.