Escherichia coli YaeJ protein mediates a novel ribosome-rescue pathway distinct from SsrA- and ArfA-mediated pathwaysmmi_

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1 Molecular Microbiology (2011) 80(3), doi: /j x First published online 21 March 2011 Escherichia coli YaeJ protein mediates a novel ribosome-rescue pathway distinct from SsrA- and ArfA-mediated pathwaysmmi_ Yuhei Chadani, 1 Katsuhiko Ono, 1 Kazuhiro Kutsukake 1,2 and Tatsuhiko Abo 1,2 * 1 Graduate School of Natural Science and Technology, Okayama University, Okayama , Japan. 2 Department of Biology, Faculty of Science, Okayama University, Okayama , Japan. Summary Accumulation of stalled ribosomes at the 3 end of mrna without a stop codon (non-stop mrna) is supposed to be toxic to bacterial cells. Escherichia coli has at least two distinct systems to rescue such stalled ribosomes: SsrA-dependent trans-translation and ArfA-dependent ribosome rescue. Combination of the ssra and arfa mutations is synthetically lethal, suggesting the significance of ribosome rescue. In this study, we identified the E. coli yaej gene, encoding a peptide-release factor homologue with GGQ motif, as a multicopy suppressor of the lethal phenotype of ssra arfa double mutant. The YaeJ protein was shown to bind to ribosomes. Both in vivo and in vitro, YaeJ showed the ribosome-rescue activity and promoted the hydrolysis of peptidyl-trna residing in the stalled ribosome. Missense mutation in the GGQ motif or deletion of the C-terminal unstructured tail abolished both the suppressor activity for ssra arfa synthetic lethality and the ribosome-rescue activity, suggesting the importance of these structural features. On the basis of these observations, we propose that YaeJ acts as a stop codon-independent peptidyl-trna hydrolysing factor through binding to ribosomes stalled at the 3 end of non-stop mrnas. It was also suggested that ArfA and YaeJ rescue the stalled ribosomes by distinct mechanisms. Introduction Translation terminates when the translating ribosome encounters an in frame stop codon. In bacteria such as Accepted 21 February, *For correspondence. tabo@cc. okayama-u.ac.jp; Tel. (+81) ; Fax (+81) Escherichia coli, class I polypeptide release factors RF1 and RF2 recognize the stop codon and bind to the A site of ribosomes, leading to the hydrolysis of the ester bond between the nascent polypeptide and trna. Then, a GTP-bound class II RF, RF3, catalyses the dissociation of class I RFs. This step requires the hydrolysis of GTP, and the GDP-bound form of RF3 falls off the ribosome because of its low affinity to the ribosome. The remaining complex composed of the ribosome, mrna, and deacylated trna will be dissociated by factors, including ribosome recycling factor (also called RF4), elongation factor-g (EF-G) and initiation factor 3 (for reviews, see Kisselev et al., 2003; Wilson, 2004; Petry et al., 2008). When mrna without the in frame stop codon (non-stop mrna) is translated, ribosomes stall at its 3 end and form non-productive complexes containing peptidyl-trna. Nonstop mrna is formed by various causes. For example, the cleavage of mrna as a regular mrna turnover process in the cell produces non-stop mrna. Inhibition of transcription also causes the production of non-stop mrna in some cases (Abo et al., 2000). Ribosome stalling in the middle of intact mrna induced by the scarcity of cognate aminoacyl-trna (Garza-Sanchez et al., 2008; Li et al., 2008) or by the action of class I RFs (Li et al., 2007) or at an inefficient stop codon (Hayes et al., 2002; Sunohara et al., 2002; 2004; Hayes and Sauer, 2003) also produces non-stop mrna by stalled ribosomespecific mrna cleavage. It has also been reported that misreading of the stop codon in the presence of suppressor trna (Ueda et al., 2002) or miscoding antibiotics (Abo et al., 2002) or inactivation of ribosome recycling factor (Hirokawa et al., 2004) allows the ribosome to reach to the 3 end of mrna and stall there. A ribosome stalled at the 3 end of non-stop mrna is not a cognate target of the ribosome-recycling pathway through the recognition of stop codon by class I RFs. In eubacteria, ribosomes stalled at the 3 end of nonstop mrnas are supposed to be rescued mainly by SsrAmediated trans-translation (for reviews, see Moore and Sauer, 2007; Keiler, 2008; Hayes and Keiler, 2010). In addition, peptidyl-trna drop-off followed by hydrolysis of peptidyl-trna may participate in ribosome rescue directly or indirectly (Singh and Varshney, 2004; Das and 2011 Blackwell Publishing Ltd

2 Ribosome rescue by E. coli YaeJ 773 Varshney, 2006). We have recently reported that E. coli ArfA (alternative ribosome-rescue factor; former YhdL) rescues the ribosomes stalled at the 3 end of non-stop mrna independently of the SsrA-mediated transtranslation (Chadani et al., 2010). ArfA is required for the growth of E. coli cells in the absence of SsrA, suggesting that accumulation of the stalled ribosome complex is toxic to bacterial cells. Both SsrA-mediated trans-translation and ArfA-dependent pathway may contribute to the dissociation of stalled ribosomes, which enables the cell to recycle ribosomes (Himeno, 2010). ArfA binds to the large subunit of ribosomes and enhances the hydrolysis of peptidyl-trna residing in the stalled ribosome, although the molecular mechanism of how it rescues ribosomes is still unknown. ArfA does not have a GGQ motif, typical catalytic residues in RFs (Frolova et al., 1999; Scarlett et al., 2003; Youngman et al., 2008), suggesting that it may cooperate with factors such as RFs or peptidyl-trna hydrolase (Pth) that hydrolyses the linkage between nascent peptide and trna. However, the possibility that ArfA itself has the hydrolysing activity should not be excluded. It is also possible that binding of ArfA induces a conformational change of ribosomes, which leads to drop-off and/or hydrolysis of peptidyl-trna. Pth is a likely candidate for this hydrolysis, but the possibility of the existence of other factors cannot be ruled out. Recently, a GGQ motif protein ICT1 was reported to act as a peptidyl-trna hydrolysing enzyme in human mitochondria (Richter et al., 2010). It is possible that such factors are also involved in the ribosome-rescue pathway in bacteria. In this study, we performed multicopy suppressor screening for the growth defect of an ssra arfa double mutant. We found that overexpression of the YaeJ protein, a putative RF and ICT1 homologue with a GGQ motif (Handa et al., 2010a), supports the growth of cells lacking SsrA and ArfA. YaeJ was shown to rescue stalled ribosomes independently of SsrA and ArfA. Comparison of the biochemical property of ArfA and YaeJ suggests that they rescue the stalled ribosome by distinct mechanisms. Biological significance of YaeJ will be also discussed. Results Suppression of the lethality of ssra arfa double mutant E. coli by a multicopy yaej gene We have recently reported that ArfA, an alternative ribosome-rescue factor, is required for the growth of SsrAdeficient E. coli cells (Chadani et al., 2010). However, the mechanism of how ArfA rescues the stalled ribosomes is still unknown. To elucidate this, we screened multicopy suppressors for the growth defect of the ssra arfa double mutant from the pstv28-based genomic library of TA331 (DssrA). CH115 (DarfA DssrA) harbouring pbad24-ssra, which expresses SsrA from the arabinose-inducible promoter, was grown in the presence of arabinose to induce SsrA expression, transformed with the library and then spread onto LB agar plates containing chloramphenicol and ampicillin but not arabinose. Most clones thus obtained had plasmids carrying the arfa gene, but one clone had a plasmid containing genes other than arfa. This plasmid, named pl-5, had a 4.5 kb HindIII fragment containing a DNA region that encompassed tils to yaek. Subcloning experiments revealed that pstv28 carrying the yaej gene, a 420 bp open reading frame (ORF) located at 4.6 min on the E. coli chromosome, suppressed the lethal phenotype of ssra arfa double mutation. Effect of YaeJ depletion on cell growth JW0187 (BW25113 yaej::frt-km r -FRT) is available among the Keio collection of NBRP: E. coli (NIG, Japan) (Baba et al., 2006), indicating that yaej is dispensable for E. coli. If the chromosomally encoded yaej gene plays an important role in ribosome rescue, the disruptant of the gene may show a translation-related phenotype as the ssra or arfa disruptant does. So, we checked the effect of yaej disruption in combination with ssra or arfa mutation. However, we found that either of the double mutants CH141 (DyaeJ DssrA) and CH161 (DyaeJ DarfA) did not show any growth defect (data not shown), indicating that the chromosomally encoded yaej gene does not play a critical role in viability of the cells. We next compared the effect of the depletion of SsrA on the growth of arfa mutant and arfa yaej double mutant cells. Cells of strain CH115 (DssrA DarfA) or CH172 (DyaeJ DssrA DarfA) harbouring pbad24-ssra were grown first in LB medium containing arabinose and then in the same medium containing glucose instead of arabinose. As shown previously (Chadani et al., 2010), growth of CH115/pBAD24-ssrA started to slow down and stopped approximately 1 h after the medium change at 37 C. CH172/pBAD24-ssrA showed similar decline in the OD 660 value, but the colony forming unit was 2 3 times lower than that of CH115/pBAD24-ssrA at every time point examined (Table S1). This indicates that the yaej gene plays a role in cell viability in the absence of SsrA and ArfA. Upon SsrA ArfA double depletion, ribosome complexes formed at the 3 end of non-stop mrna accumulate in the cells (Chadani et al., 2010), which is supposed to be causative of the growth defect in the ssra arfa double mutant. Chromosomally encoded YaeJ may rescue such ribosomes to the extent that it partially, but not fully, recovers cell viability. Only when overexpressed, it supports the growth of ssra arfa double mutant. Taken together, we conclude YaeJ has the ribosome-rescue activity.

3 774 Y. Chadani, K. Ono, K. Kutsukake and T. Abo A Fig. 1. A. Alignment of the relevant region of E. coli YaeJ, RF1 (PrfA), and RF2 (PrfB) and the C-terminal unstructured tail of SmpB. Conserved domains and motifs are shown by boxes. The C-terminal unstructured tail of YaeJ is underlined. Basic residues within the unstructured tail region of YaeJ and SmpB are shown by bold letters. B. Suppression of synthetic lethal phenotype of ssra and arfa mutations by plasmid-borne His 6-YaeJ. CH111/pBAD33-ssrA harbouring pqe80l (vector), pch400 (YaeJ(wt)), pch401 (YaeJ-dC20), pch402 (YaeJ-dC40), pch403 (YaeJ-dC60) or pch410 (YaeJ-G27A) was streaked onto LB agar plates containing arabinose (top, left), glucose (top, right) or glucose and 100 mm of IPTG (bottom, left) and incubated for 20 h at 37 C. B Mutational analysis of YaeJ YaeJ protein, which is 140 amino acids in length, shares significant homology with domain III, but not with domain II, of E. coli RF1 and RF2 (Fig. 1A). Domain III of RF1 and RF2 contains a GGQ motif, which is required for their peptidyl-trna hydrolysing activity (Frolova et al., 1999; Scarlett et al., 2003; Youngman et al., 2008). Domain II contains a tripeptide motif PAT (RF1) or SPF (RF2), which is involved in the recognition of stop codons (Ito et al.,

4 Ribosome rescue by E. coli YaeJ ; Scarlett et al., 2003; Laurberg et al., 2008). These suggest that YaeJ functions like a release factor but in a codon-independent manner. To see if the GGQ motif of YaeJ is important for its suppressor activity, we introduced a mutation into the yaej ORF to change its GGQ sequence to GAQ. N-terminally His 6-tagged YaeJ (His 6-YaeJ) produced from pch400 supported the growth of SsrA ArfA double depleted CH111 (DarfA DssrA) cells, indicating that overexpressed His 6-YaeJ can suppress the lethal phenotype of the ssra arfa double mutant. His 6-YaeJ-G27A, the mutated version of His 6-YaeJ having GAQ sequence instead of GGQ sequence, did not suppress the lethal phenotype of the ssra arfa double mutant, indicating that the GGQ motif of YaeJ is crucial for suppression of the lethality of the ssra arfa double mutant (Fig. 1B). Another intriguing feature of YaeJ is its C-terminal unstructured tail. NMR analysis of Salmonella YaeJ (available in the database; PDB 2jy9) predicted that YaeJ has an unstructured tail spanning approximately 40 amino acid residues at its C-terminus. SmpB, a specific protein partner of SsrA, which is essential for transtranslation, also has an unstructured C-terminal tail. SmpB was shown to bind to the ribosomes during movement of the mrna module of tmrna through ribosomes (Shpanchenko et al., 2005). It was also hypothesized that the C-terminal tail of SmpB occupies the mrna path within the ribosome in the early stage of transtranslation (Jacob et al., 2005; Sundermeier et al., 2005; Kurita et al., 2007; 2010). C-terminal regions of SmpB and YaeJ are both rich in basic amino acid residues such as lysine and arginine (Fig. 1A), raising the possibility that the C-terminal tail of YaeJ is also important for its activity. To evaluate the significance of the C-terminal unstructured tail, we constructed plasmids producing a series of truncated His 6-YaeJ that lack C-terminal 20, 40 and 60 amino acid residues, which are His 6-YaeJ-dC20, His 6-YaeJ-dC40, and His 6-YaeJ-dC60 respectively. When assayed on LB agar plates, none of these C-terminally truncated His 6-YaeJ mutants supported the growth of the SsrA ArfA double depleted CH111 cells (Fig. 1B). So, we conclude that the C-terminal tail of YaeJ is required for its activity to support the growth of SsrA ArfA double depleted cells in vivo. Association of YaeJ with ribosomes If YaeJ functions as a codon-independent RF, it should bind to ribosomes. To see this, we performed fractionation studies using various His 6-YaeJ proteins, which were detected by anti-his 6 Western blotting. E. coli CH131 cell lysate containing His 6-YaeJ was fractionated by centrifugation, and distribution of His 6-YaeJ was analysed by anti-his 6 Western blotting (Fig. 2). The wild-type His 6-YaeJ Fig. 2. Association of His 6-YaeJ and its variants to ribosomes. S30 lysate prepared from CH131 harbouring pqe80l (-), pch400 (wt), pch410 (G27A), pch401 (dc20), pch402 (dc40) or pch403 (dc60) was fractionated into S100 and P100 fractions by centrifugation and analysed by anti-his 6 Western blotting. The cleavage product of His 6-YaeJ or His 6-YaeJ-G27A is indicated by asterisks. protein was preferentially fractionated into the ribosomecontaining P100 fraction, but a substantial amount of His 6-YaeJ was also detected in the S100 fraction. His 6-YaeJ-G27A was also preferentially fractionated into the P100 fraction. These results suggest that YaeJ binds to ribosomes, and this binding does not require the GGQ motif of YaeJ. Presence of YaeJ in the S100 fraction may be due to the excess amount of YaeJ in the lysate. The amount of the C-terminally truncated versions, His 6-YaeJ-dC20 and His 6-YaeJ-dC40, in the S30 fraction was as much as that of full-length His 6-YaeJ (Fig. 2, top panel). However, they were more abundant in the S100 fraction than in the P100 fraction (Fig. 2, middle and lower panels). These results suggest that the C-terminal region of His 6-YaeJ is important for its ribosome binding, but not for its expression or stability. The amount of His 6-YaeJ-dC60 was much lower than other His 6-YaeJ variants, indicating that deletion of the C-terminal 60

5 776 Y. Chadani, K. Ono, K. Kutsukake and T. Abo Fig. 3. Effect of YaeJ depletion in various genetic backgrounds on the expression profiles of CRP-ST and CRP-NST in vivo. A. Schematic drawing of the genes for CRP-ST and CRP-NST carried on pch326 and pch336 respectively. The nucleotide sequence of pch326 and pch336 is identical except that the crp gene on pch326 has an in frame stop codon upstream to the trpa terminator, whereas that on pch336 does not. The crp ORF is shown by an open box, where His 6-tag regions are filled. T5 promoter (P T5) and trpa terminator (T trpa) are also shown. Filled triangles indicate in frame stop codons. Artificially introduced SsrA-tag sequence region is indicated by a hatched box. B and C. Profile of CRP-ST (B) or CRP-NST (C) in the wild type, DssrA, DarfA, DyaeJ, DssrA DyaeJ and DarfA DyaeJ backgrounds, as indicated above the lanes. Profile of CRP-NST in ssra DD, ssra DD DarfA, ssra DD DyaeJ and ssra DD DarfA DyaeJ backgrounds is also shown in panel C. TA341 (W3110 Dcrp, lane 1), TA501 (TA341 DssrA, lane 2), CH231 (TA341 DarfA, lane 3), CH251 (TA341 DyaeJ, lane 4), CH261 (TA501 DyaeJ, lane 5), CH271 (CH251 DarfA, lane 6), TA481 (TA341 ssra DD, lane 7), CH281 (TA481 DarfA, lane 8), CH291 (TA481 DyaeJ, lane 9) or CH301 (CH281 DyaeJ, lane 10) harbouring pch326 (B) or pch336 (C) were cultured in LB until the culture reached the mid-log phase. Then, expression of CRP-ST or CRP-NST was induced by 250 mm of IPTG for 1 h, and the samples were analysed by Western blotting using anti His 6-tag antibody. Bands for CRP-ST, CRP-NST-DD and CRP-NST are indicated by arrows. amino acid residues lowers the expression level or stability of His 6-YaeJ. CH131 cells expressing full-length or G27A mutant His 6-YaeJ contained a smaller molecule detected by anti-his 6 antibody (Fig. 2, asterisks), which will be discussed later. Effect of YaeJ depletion on the expression of a model protein Next, we asked if the depletion of YaeJ has any effect on the translation of model mrna with or without the stop codon. To see this, we used N-terminally His 6-tagged E. coli camp receptor protein (CRP) variants, CRP-NST and CRP-ST, as model proteins (Fig. 3A). CRP-NST is a modified CRP protein produced by pch336. Its mrna does not have an in frame stop codon because the transcription of the gene for CRP-NST terminates at the trpa terminator inserted within the ORF. Transcriptional readthrough at the trpa terminator of pch336 may result in the production of longer mrna, but the protein produced from the readthrough mrna will be degraded because it has the SsrA tag sequence at its C-terminus. This enables us to detect only the CRP-NST protein produced from non-stop mrna. On the other hand, pch326 produces CRP-ST and served as a control whose mrna has a stop codon. pch326 and pch336 were introduced into various strains by transformation and the resulting transformant cells were grown in LB medium. Then, the expression of CRP-ST and CRP-NST was analysed by anti-his 6 Western blotting. The expression levels of CRP-ST were almost the same in all the strains examined, suggesting that disruption of yaej does not affect the expression of the protein encoded by mrna having a stop codon (Fig. 3B). On the other hand, CRP-NST was detectable only when SsrA was absent (Fig. 3C, lanes 2 and 5) or replaced by SsrA DD, an SsrA variant that attaches a proteolysis-resistant tag instead of the SsrA tag (Fig. 3C, lanes 7 10). In the absence of SsrA, disruption of yaej caused a subtle decrease in the amount of CRP-NST (Fig. 3C, lanes 2 and 5). In the presence of SsrA DD, the

6 Ribosome rescue by E. coli YaeJ 777 Fig. 4. Effect of overexpression of His 6-YaeJ, His 6-ArfA, and their variants on the expression profile of CRP-NST in vivo. TA341 harbouring pch336 together with pqe80l (empty vector, lane 1), pch201 (His 6-ArfA, lane 2), pch221 (His 6-ArfA-A18T, lane 3), pch400 (His 6-YaeJ, lane 4), pch410 (His 6-YaeJ-G27A, lane 5), pch401 (His 6-YaeJ-dC20, lane 6), pch402 (His 6-YaeJ-dC40, lane 7) or pch403 (His 6-YaeJ-dC60, lane 8) was cultured in LB until the culture reached the mid-log phase. Then, the expression of CRP-NST was induced by 500 mm of IPTG for 2 h, and samples were analysed by Western blotting using anti His 6-tag antibody. The band for CRP-NST is indicated by an arrow. The bands for His 6-tagged ArfA, YaeJ, and their variants are indicated by brackets. Longer exposure of lane 8 is also shown (lane 8 ). CRP-NST band was clearly detected when ArfA was present. In the absence of ArfA, this band was faintly detected when YaeJ was present, but could not be detected in the absence of YaeJ (Fig. 3C, lanes 7 10). These results suggest that ribosome rescue by chromosomally encoded YaeJ is less efficient than that by the ArfA or SsrA system, and neither of these systems requires the chromosomally encoded YaeJ protein. YaeJ-dependent rescue of ribosomes stalled at the end of a model non-stop mrna If the suppression of lethality of the ssra arfa double mutant by overexpressed YaeJ is caused by its codonindependent peptidyl-trna hydrolysing activity, YaeJ may compete with the SsrA system on the ribosome stalled at the 3 end of the non-stop mrna and release the nascent polypeptide, as ArfA does. To test this, we analysed the effect of the overexpression of N-terminally His 6- tagged YaeJ upon the expression profile of CRP-NST in vivo. CRP-NST was not detectable when expressed in the TA341 (Dcrp) cells (Fig. 4, lane 1), indicating that CRP- NST produced from non-stop mrna is degraded as a result of trans-translation. Then, we introduced the plasmids that overexpress N-terminally His 6-tagged ArfA, ArfA-A18T or YaeJ into TA341/pCH336 by transformation and analysed the expression profile of CRP-NST by anti-his 6 Western blotting. As we showed previously (Chadani et al., 2010), CRP-NST was detected when His 6-ArfA, but not His 6-ArfA-A18T, was overexpressed, indicating that ArfA releases the ribosome stalled at the 3 end of the non-stop mrna for CRP-NST (Fig. 4, lanes 2 and 3). CRP-NST was detected when His 6-YaeJ was overexpressed (Fig. 4, lane 4), indicating that YaeJ competes with the SsrA system for a ribosome stalled at the 3 end of the non-stop mrna, as ArfA does. The possibility that YaeJ rescues another set of stalled ribosomes than that rescued by the SsrA system cannot be ruled out. We also introduced plasmids that overexpress mutant versions of N-terminally His 6-tagged YaeJ, namely His 6-YaeJ-G27A, YaeJ-dC20, YaeJ-dC40 and YaeJ-dC60, into TA341/pCH336 and analysed the expression profile of CRP-NST by anti-his 6 Western blotting. Overexpression of these loss-of-function mutant versions did not result in the increased amount of CRP-NST (Fig. 4, lanes 5 8). The peptidyl-trna hydrolysing activity of YaeJ was exerted independently of the chromosomal arfa gene (Fig. S1). This indicates that ArfA and YaeJ independently act to rescue the ribosome stalled at the 3 end of the non-stop mrna. Resolution of peptidyl-trna promoted by YaeJ in vitro Ribosome-rescue activity of YaeJ was analysed by using S30 in vitro translation system. The S30 lysate prepared from CH161 (DarfA DyaeJ) was treated with anti-ssra oligonucleotide to inhibit the activity of SsrA (Hanes and Plückthun, 1997; Chadani et al., 2010). Using this lysate, translation of in vitro prepared non-stop mrna was performed. After treatment with purified proteins or puromycin, samples were separated by Bis-Tris SDS-PAGE under the neutral condition, and the synthesized proteins were visualized by anti-his 6 Western blotting. The same samples were also analysed by Northern blotting using crp probe. Even without any treatment, the CRP-NST band was detected along with the peptidyl-trna band (CRP-NST-tRNA), maybe reflecting the spontaneous drop-off and subsequent hydrolysis of peptidyl-trna in the reaction (Fig. 5A, lane 1). As reported previously (Chadani et al., 2010), treatment with His 6-ArfA resulted in

7 778 Y. Chadani, K. Ono, K. Kutsukake and T. Abo A B Fig. 5. Resolution of peptidyl-trna (CRP-NST-tRNA) by His 6-ArfA, His 6-YaeJ and puromycin in vitro. A. In vitro prepared non-stop mrna for CRP-NST was translated in E. coli CH161 (DarfA DyaeJ) S30 extract supplemented with the anti-ssra oligonucleotide. After 30 min translation at 37 C, 2 mg ml -1 kasugamycin was added to inhibit further initiation of translation. To the reaction mixtures, 100 nm of purified His 6-ArfA (ArfA, lane 2), His 6-ArfA-A18T (A18T, lane 3), His 6-YaeJ (YaeJ, lane 4), His 6-YaeJ-G27A (G27A, lane 5) or 10 mm of puromycin (Pm, lane 6) was added and incubation was continued for another 10 min at 37 C. Samples were then analysed by Bis-Tris SDS-PAGE and Western blotting using anti-his 6 antibody. The same samples were also analysed by Northern blotting using a crp probe or 5S rrna probe. The protein bands corresponding to the CRP-NST-tRNA and CRP-NST are indicated by arrows. B. One hundred and fifty microlitres of reaction mixture treated with 200 nm of His 6-YaeJ or His 6-YaeJ-G27A as panel A were analysed by sucrose gradient ultracentrifugation after addition of 200 mg ml -1 chloramphenicol. Distribution of CRP-NST-tRNA, CRP-NST, His 6-YaeJ or His 6-YaeJ-G27A was analysed by Western blotting using anti-his 6 antibody. Distribution of the mrna for CRP-NST was also analysed by Northern blotting using a crp probe. Bands corresponding to the CRP-NST-tRNA, CRP-NST, His 6-YaeJ and His 6-YaeJ-G27A are indicated by arrows. the disappearance of peptidyl-trna band and reduction of non-stop model mrna, whereas treatment with His 6-ArfA-A18T had no effect, indicating that ArfA, but not ArfA-A18T, promotes the resolution of peptidyl-trna (Fig. 5A, lanes 2 and 3). When the sample was treated with purified His 6-YaeJ, the peptidyl-trna band disappeared, and the amount of non-stop model mrna reduced, indicating that YaeJ also promotes the resolution of peptidyl-trna (Fig. 5A, lane 4). As expected, His 6-YaeJ-G27A did not have such activity, strongly indicating that the GGQ motif of YaeJ is crucial in its peptidyltrna hydrolysing activity (Fig. 5A, lane 5). Treatment with puromycin, performed as a positive control for the resolution of peptidyl-trna, resulted in the disappearance of peptidyl-trna band and reduction of the non-stop model mrna (Fig. 5A, lane 6). The same in vitro translation samples, as shown in Fig. 5A, lanes 4 and 5, were further analysed by sucrose gradient ultracentrifugation. As shown in Fig. 5B, peptidyltrna associated with ribosomes (seen as the bands indicated by an arrow labelled CRP-NST-tRNA ) disappeared when His 6-YaeJ, but not His 6-YaeJ-G27A, was present. On the other hand, the level of CRP-NST polypeptide (seen as the bands indicated by an arrow labelled CRP-NST ) in the ribosome-free fraction was much higher when His 6-YaeJ (Fig. 5B, left), but not His 6-YaeJ-G27A (Fig. 5B, right), was added to the reaction. This result indicates that ribosomes stalled at the end of the non-stop mrna and accumulated upon SsrA and ArfA double depletion were rescued by His 6-YaeJ. Both His 6-YaeJ and His 6-YaeJ-G27A were detected in the ribosome-containing fraction, indicating that YaeJ binds to ribosomes to promote peptidyl-trna

8 Ribosome rescue by E. coli YaeJ 779 Fig. 6. Stability of peptidyl-trna in S30 lysate. In vitro prepared non-stop mrna for CRP-NST was translated in S30 lysate prepared from CH101 (DarfA) or CH161 (DarfA DyaeJ) supplemented with anti-ssra oligonucleotide. Translation reaction was stopped by adding 2 mg ml -1 kasugamycin, and the samples were taken from the reaction mixture at the time points indicated. Samples were then analysed by Bis-Tris SDS-PAGE and anti-his 6 Western blotting. The bands for CRP-NST-tRNA and CRP-NST are indicated by arrows. Band density was evaluated using Multi Gauge Ver. 3.0 (FUJIFILM), plotted as shown below the gel, and the half-life of peptidyl-trna in each lysate was calculated. The most representative result of six independent assays is shown. hydrolysis, and the G27A mutation does not affect its affinity to ribosomes. This result also indicates that most of the chromosomally encoded YaeJ proteins are bound to ribosomes, although substantial amount of His 6-YaeJ was left unbound to ribosomes when excess amount of His 6-YaeJ was present, as seen in Fig. 2. Purified His 6-YaeJ has been shown to promote the resolution of peptidyl-trna residing in the stalled ribosomes, but chromosomally encoded YaeJ cannot support the cell viability in the absence of both SsrA and ArfA. This may be due to the weak activity or scarcity of YaeJ. We then checked this hypothesis. If chromosomally encoded YaeJ acts as a ribosome-rescue factor, the absence of YaeJ may increase the stability of peptidyl-trna. To see this, we compared the stability of peptidyl-trna produced from CRP-NST mrna in the S30 lysates prepared from CH101 (DarfA) and CH161 (DarfA DyaeJ). As shown in Fig. 6, peptidyl-trna was more stable in the S30 lysate prepared from CH161 (half-life, min) than that of CH101 (halflife, 5.15 min), indicating that chromosomally encoded YaeJ has a ribosome-rescue function, although it is not strong enough to support the viability of ssra arfa double mutant cells. YaeJ and ArfA rescue the stalled ribosome by distinct mechanisms To analyse the mechanisms by which YaeJ rescues stalled ribosomes, we isolated the stalled ribosome complexes from S30 translation reaction mixture using Ni-affinity resin. The isolated ribosome-nascent peptide complex was shown to contain peptidyl-trna (CRP-NST-tRNA) (Fig. 7, lane 1). The isolated complex was then treated with the purified preparation of His 6-YaeJ or His 6-ArfA or various concentrations of puromycin. Samples were separated by Bis-Tris SDS-PAGE, and then proteins were visualized by anti-his 6 Western blotting. Fig. 7. Resolution of peptidyl-trna residing in an isolated ribosome complex in vitro. In vitro prepared non-stop mrna for CRP-NST was translated in E. coli CH161 S30 extract. From the reaction mixture, the ribosome complex containing CRP-NST-tRNA and its mrna was isolated using Ni-chelating resin, as described in Experimental procedures. The isolated complex was then treated with 200 nm of purified His 6-ArfA (lane 2), His 6-YaeJ (lane 3) or 1 mm (lane 4), 10 mm (lane 5) or 100 mm (lane 6) of puromycin for 20 min at 37 C. Lane 1 is the control, where the isolated complex was incubated without treatment. The sample was analysed by Bis-Tris SDS-PAGE and anti-his 6 Western blotting. The bands for CRP-NST-tRNA, CRP-NST, His 6-YaeJ and His 6-ArfA are indicated by arrows.

9 780 Y. Chadani, K. Ono, K. Kutsukake and T. Abo When the complex was treated with His 6-YaeJ, peptidyltrna almost completely disappeared (Fig. 7, lane 3), indicating that the stalled ribosome was rescued. This result suggests that YaeJ does not require a cellular factor to rescue the stalled ribosome. On the other hand, when the complex was treated with His 6-ArfA, peptidyl-trna remained detectable (Fig. 7, lane 2). We reported previously that when added to the S30 translation reaction, His 6-ArfAshowed ribosome-rescue activity (Chadani et al., 2010). This difference may be attributed to a cellular factor, which was present in S30 lysate but absent from the isolated ribosome-complex fraction. In other words, ArfA requires a cellular factor to rescue the stalled ribosome. The function of this unknown factor may include the enhancement of ribosome binding of ArfA, enhancement of peptidyl-trna drop-off by ArfA or enhancement of peptidyltrna hydrolysis by ArfA. Addition of puromycin resulted in resolution of the peptidyl-trna in a manner dependent on its concentration (Fig. 7, lanes 4 6), indicating that peptidyl-trna detected here resides in ribosomes. Discussion Recent studies have shown that bacteria have at least two rescue systems for ribosomes stalled at the end of the non-stop mrna, SsrA-mediated trans-translation (Keiler et al., 1996) and ArfA-mediated ribosome-rescue system (Chadani et al., 2010). The SsrA system is widely distributed among eubacteria (for reviews, see Moore and Sauer, 2007; Keiler, 2008; Hayes and Keiler, 2010), whereas homologues of E. coli ArfA are found only in enterobacteria such as E. coli and Salmonella, but not in other bacterial species (Chadani et al., 2010). Here, we have identified YaeJ as a multicopy suppressor for the lethal phenotype of arfa ssra double mutant E. coli cells. Both in vivo and in vitro studies revealed that YaeJ binds to ribosomes and possesses a ribosomerescue activity. Very recently, the activity of YaeJ to hydrolyse peptidyl-trna on stalled ribosomes was reported by Handa et al. (2010b). Their data and our results shown here strongly suggest that YaeJ is the third ribosomerescue factor, although it supports arfa ssra double mutant E. coli cells only when overexpressed. YaeJ is distributed among a wide range of eubacteria (Handa et al., 2010b). It has the GGQ motif, which is typical to RFs (Frolova et al., 1999; Scarlett et al., 2003; Youngman et al., 2008), but it does not have homology to domain II of RFs, which contains the codon-recognizing motif (Ito et al., 2000; Scarlett et al., 2003; Laurberg et al., 2008). The GGQ motif of YaeJ is important for its ribosome-rescue activity, confirming the idea that YaeJ has the hydrolysing activity that cleaves the peptide-trna bonding. Lack of the homology to domain II of RFs is consistent with the proposed activity of YaeJ as a codon-independent ribosome-rescue factor. Instead of the codon-recognizing motif, YaeJ has an unstructured tail at its C-terminus, which is required for its ribosome binding. SmpB, a specific protein partner of SsrA essential for trans-translation, also has a C-terminal tail that is predicted to be unstructured by NMR or X-ray diffraction analyses (Dong et al., 2002; Gutmann et al., 2003; Someya et al., 2003). By intensive chemical probing and mutational studies along with structural analyses, the role of the C-terminal tail of SmpB in trans-translation has been proposed to promote the early stage of trans-translation (Sundermeier et al., 2005; Kurita et al., 2010). The C- terminal tail of A-site-bound SmpB is positioned at close proximity to the decoding centre (Kurita et al., 2007; 2010; Nonin-Lecomte et al., 2009), possibly by mimicking mrna. The occupation of the mrna path within the ribosome by the C-terminal tail was discussed to be important for the accommodation of the SmpB-SsrA complex into the A-site of ribosomes (Kurita et al., 2007; 2010; Nonin- Lecomte et al., 2009). However, the feature of the C- terminal tail of YaeJ seems to be somehow different from that of SmpB. C-terminal truncation of SmpB did not abolish the ribosome binding of the SmpB-SsrA complex (Sundermeier et al., 2005), whereas C-terminal truncation reduced the ribosome binding of YaeJ, suggesting that the C-terminal region of YaeJ is important for ribosome binding of YaeJ. In the lysate of CH131 cells expressing full-length or G27A mutant His 6-YaeJ, we observed a smaller molecule detected by anti-his 6 antibody (Fig. 2, asterisks). We suspect this is the result of cleavage of these proteins during the fractionation procedure. Although we do not know what is the factor(s) responsible for this cleavage, the cleavage site or the important structure for this cleavage must reside around the 120th amino acid residue, because the smaller protein product in lanes 2 and 3 showed a similar migration rate as His 6-YaeJ-dC20, and such a small protein product was not seen in the sample obtained from CH131 expressing His 6-YaeJ-dC20. YaeJ promotes hydrolysis of peptidyl-trna within isolated stalled ribosome complexes, indicating that all the factors required for YaeJ-mediated peptidyl-trna hydrolysis are present in the stalled ribosome complexes. Importance of the GGQ motif of YaeJ in its ribosome-rescue function suggests that this motif is crucial for peptidyltrna hydrolysis, as that of class I RF. If the GGQ motif of YaeJ is involved in hydrolysis of peptidyl-trna, it should be positioned precisely at the peptidyl transferase centre. We speculate the C-terminal tail of YaeJ also resides in the mrna path within ribosomes as that of SmpB and positively charged amino acid residues within this region facilitate precise positioning of YaeJ in ribosomes. It is intriguing if the occupation of the mrna path by the unstructured peptide chain is a general feature for codonindependent ribosome-rescue factors. Mutational study of

10 Ribosome rescue by E. coli YaeJ 781 the C-terminal tail region and its comparison with other GGQ proteins are our next subjects. Comparison of the activity of YaeJ and ArfA also provides us with several intriguing aspects concerning the mechanism of ribosome rescue by ArfA. Although both YaeJ and ArfA rescue stalled ribosomes, their actions on isolated ribosome complexes are different. YaeJ resolves the peptidyl-trna by itself, whereas ArfA does not. This indicates that ArfA requires a cofactor that is absent in the isolated ribosome complex preparation. There are many candidates for this missing factor. ArfA may need some factor(s) to get into the stalled ribosome, as trna requires EF-Tu or SsrA requires SmpB and EF-Tu. Alternatively, ArfA may just help some other factor(s) to rescue the ribosome. In this case, Pth and class I RFs are the most possible candidates for the factor that cooperates with ArfA, but participation of other factors should be also considered. ArfA does not seem to have a C-terminal tail equivalent to that of YaeJ or SmpB. It is interesting whether ArfA, like the C-terminal tail of SmpB or YaeJ, functions to support the precise positioning of the factor cooperating with ArfA. In this point of view, it is noteworthy that the central region of E. coli ArfA is rich in basic amino acids and contains aromatic amino acids as the C-terminal region of SmpB and YaeJ does. We are now trying to identify the factor required for ArfA-dependent ribosome rescue. Identification of such factors may help us understand further the molecular mechanism of ArfAdependent ribosome rescue. What is the physiological role of YaeJ in E. coli cells? Our study clearly showed that chromosomally encoded YaeJ cannot support the growth of SsrA ArfA double depleted cells, and depletion of YaeJ from the cells in various backgrounds had almost no effect on the cell viability or translation of mrna with or without a stop codon. However, analysis on the stability of peptidyl-trna in vitro showed that chromosomally encoded YaeJ enhances the hydrolysis of peptidyl-trna. YaeJ could support the growth of the cell lacking both SsrA and ArfA only upon overexpression. The effect of yaej disruption on the cell viability was only seen in the absence of both SsrA and ArfA. From these results, we assume that the activity of chromosomally encoded YaeJ is not high enough to decrease the stalled ribosomes to the level required for cells to survive. Under natural conditions, double mutation in ssra and arfa might be rare. A rather possible condition is that SsrA and ArfA activities are somehow lowered or titred out by excess amounts of non-stop mrna accumulated in the cell, e.g. under severe nutrition starvation (Garza-Sanchez et al., 2008; Li et al., 2008). In such situations, ribosome-rescue activity of YaeJ, although very low in efficiency, should help in dissociation of the stalled ribosome complex. Considering its activity, we propose here to rename yaej arfb for alternative ribosome-rescue factor B. SsrA, ArfA, YaeJ and possibly Pth use different mechanisms to rescue ribosomes. They seem to be redundant at the first glance, but there must be a rationale for the cell to have these redundant systems to rescue ribosomes. First of all, ribosome stalling may be a much deleterious incident within the cell than has been thought, and the stalled ribosome should be rescued by any means. It is also possible that the bacterial cell uses appropriate systems depending on the situation to rescue the ribosome. For example, SsrA-mediated trans-translation, the major ribosome-rescue system in the cell, is accompanied by energy-consuming proteolysis. This enables the cell to utilize nascent polypeptides as a source of amino acids. However, in the situation where energy exhaustion is also a serious problem, systems other than trans-translation may be advantageous. Experimental procedures E. coli strains, phages, plasmids and primers The E. coli strains used in this study are listed in Table 1. Phage P1-mediated transduction (Miller, 1992) was used to introduce the yaej mutation from JW0187 to appropriate strains. Removal of the FRT-Km r -FRT cassette from the chromosome of the transductant was performed using pcp20, as described by Datsenko and Wanner (2000). The plasmids and primers used in this study are listed in Tables 2 and 3 respectively. A 490 bp DNA fragment containing yaej was PCR amplified using the primer pair JF01_EcoRI and JR01_SalI and pl-5 as a template, digested with EcoRI and SalI, and cloned into the corresponding site of pstv28 to obtain pstv-yaej. A 136 bp DNA fragment PCR amplified from pstv-yaej by using the primer pair JF01_EcoRI and G27A_rv and a 374 bp DNA fragment PCR amplified from the same plasmid by using the primer pair JR01_SalI and G27A_fw were mixed and used as partially overlapping templates for the PCR reaction using the primer pair JF01_EcoRI and JR01_SalI. The resulting 490 bp DNA fragment was digested with EcoRI and SalI, and cloned into the corresponding site of pstv28 to obtain pstv-yaej- G27A. A 452 bp DNA fragment containing yaej was PCR amplified using the primer pair JF02_BamHI and JR01_SalI and pstv-yaej as a template, digested with BamHI and SalI, and cloned into the corresponding site of pqe80l to construct pch400. pch401, pch402 and pch403 were constructed by cloning BamHI- and HindIII-digested DNA fragments that were PCR amplified using the primer pairs JF02_BamHI and yaej_dc20-hindiii_rv, JF02_BamHI and yaej_dc40- HindIII_rv, and JF02_BamHI and yaej_dc60-hindiii_rv, respectively, and pch400 was used as a template into the BamHI-HindIII region of pqe80l. A 452 bp DNA fragment containing yaej having a G27A mutation was PCR amplified using primer pair JF02_BamHI and JR01_SalI and pstvyaej-g27a as a template, digested with BamHI and SalI, and cloned into the corresponding site of pqe80l to construct pch410. A 51 bp DNA fragment containing the SsrA-tag coding sequence was prepared by PCR using mutually annealing primers, degradation tag-fw and degradation tag-rv,

11 782 Y. Chadani, K. Ono, K. Kutsukake and T. Abo Table 1. Strains used. Strain Description Source/reference BL21 F -, ompt, hsds (r B- m B- ), gal, dcm Laboratory stock W3110 F -, l -, IN(rrnD-rrnE)1, rph-1 Laboratory stock JW0187 BW25113 DyaeJ::FRT-Km r -FRT Baba et al. (2006) NBRC, NIG, Japan TA331 W3110 DssrA::FRT Abo et al. (2002) TA341 W3110 Dcrp::FRT Abo et al. (2002) TA481 W3110 ssra DD -FRT Dcrp::FRT Sunohara et al. (2002) TA501 W3110 DssrA::FRT Dcrp::FRT Abo et al. (2002) CH101 W3110 DarfA::FRT Chadani et al. (2010) CH111 W3110 DarfA::FRT DssrA::FRT This study CH115 W3110 DarfA::FRT DssrA::FRT laci3 laczdm211 lacy + zai::tn10 Chadani et al. (2010) CH131 W3110 DyaeJ::FRT This study CH141 W3110 DssrA::FRT DyaeJ::FRT This study CH161 W3110 DarfA::FRT DyaeJ::FRT This study CH172 W3110 DarfA::FRT DssrA::FRT DyaeJ::FRT laci3 laczdm211 lacy + zai::tn10 This study CH231 TA341 DarfA::FRT Chadani et al. (2010) CH251 TA341 DyaeJ::FRT This study CH261 TA341 DssrA::FRT DyaeJ::FRT This study CH271 TA341 DarfA::FRT DyaeJ::FRT This study CH281 TA481 DarfA::FRT Chadani et al. (2010) CH291 TA481 DyaeJ::FRT This study CH301 TA481 DarfA::FRT DyaeJ::FRT This study and was digested with PstI. This DNA fragment was ligated with pch322 DNA or pch332 DNA, which had been digested with NheI, Klenow filled-in, and digested with PstI to construct pch326 or pch336 respectively. To construct pbad33-ssra, a 540 bp DNA fragment containing the ssra gene was PCR amplified using a primer pair 5-ssrA-EcoRI and ssra-r and pbad24-ssra as a template, digested with EcoRI and HindIII, Klenow filled-in, and then cloned into the SmaI site of pbad33 (Guzman et al., 1995) so that the ssra gene is expressed from the arabinose promoter. Bacterial growth Escherichia coli cells were grown in LB at 37 C, unless otherwise noted. LB containing 1.5% (w/v) agar was used to prepare the culture plate. Arabinose or glucose was added to Table 2. Plasmids used. Plasmid Description Source/reference pcp20 FLP expressing plasmid Datsenko and Wanner (2000) pspt18 ColE1 derivative carrying phage polymerase expression system, Amp r Boehringer pha7 pbr322 derivative carrying crp Aiba et al. (1982) prt18 derivative of pspt18 carrying 5S rrna gene Laboratory stock pqe80l ColE1 derivative carrying His 6-protein expression system, laci q, Amp r Qiagen pbad24-ssra derivative of pbad24 carrying ssra Ono et al. (2009) pbad33 p15a derived cloning vector carrying P ara expression system, Cm r Guzman et al. (1995) pbad33-ssra derivative of pbad33 carrying ssra This study pstv28 p15a derived cloning vector, Cm r TAKARA pstv-yaej derivative of pstv28 expressing YaeJ This study pstv-yaej-g27a derivative of pstv28 expressing YaeJ-G27A This study pch102 p15a derivative carrying His 6-protein expression system, laci q,km r Chadani et al. (2010) pch201 derivative of pqe80l expressing His 6-ArfA (DC12) Chadani et al. (2010) pch221 derivative of pqe80l expressing His 6-ArfA (A18T, DC12) Chadani et al. (2010) pch322 derivative of pch102 expressing His 6-CRP (with stop codon) Chadani et al. (2010) pch326 derivative of pch102 expressing His 6-CRP (with stop codon) This study pch332 derivative of pch102 expressing His 6-CRP (without stop codon) Chadani et al. (2010) pch336 derivative of pch102 expressing His 6-CRP (without stop codon) This study pch341 derivative of pqe80l expressing His 6-CRP-FLAG Chadani et al. (2010) pch353 derivative of pspt18 expressing His 6-CRP-FLAG (without stop codon) Chadani et al. (2010) pch400 derivative of pqe80l expressing His 6-YaeJ This study pch401 derivative of pqe80l expressing His 6-YaeJ(DC20) This study pch402 derivative of pqe80l expressing His 6-YaeJ(DC40) This study pch403 derivative of pqe80l expressing His 6-YaeJ(DC60) This study pch410 derivative of pqe80l expressing His 6-YaeJ (G27A) This study

12 Ribosome rescue by E. coli YaeJ 783 Table 3. Primers used. Primer Nucleotide sequence (5-3 ) JF01_EcoRI JF02_BamHI JR01_SalI yaej_dc20-hindiii_rv yaej_dc40-hindiii_rv yaej_dc60-hindiii_rv G27A_fw G27A_rv CF01_BamHI CR01_SphI 5-ssrA-EcoRI ssra_r degradation tag_fw degradation tag-rv GGG AAT TCT CTG GAA GTG AAC CCG GAT CCA TTG TGA TTT CCC GA TTG TCG ACC ATC CAT TCC TT TTA AGC TTA TGC CAG CCT GCG CTC CCA AGC TTA TGT TGT TAA TTC TTT CCA AGC TTA CTG ACT GCG GTA TTC CGC GGG CGC ACA GCA TGT TAA TAA G TTA ACA TGC TGT GCG CCC GCG CCC T AAG GAT CCG TGC TTG GCA AAC CG ATG CAT GCG TGC CGT AAA CGA C CCG GAA TTC TAC CTT TAC ACA TTG GGG C CCC AAG CTT AAT GGG CCT AAA AGG TTC GG CCC TGC AGA GCT GCA AAC GAC GAA AAC T CCA AGC TTA AGC TGC TAA AGC GTA GTT TTC the media to the final concentration of 0.2% (w/v) or 0.4% (w/v) respectively. Appropriate antibiotics were added to the media. Bacterial growth in liquid medium was monitored by measuring the OD 660. Fractionation of cellular components Crude lysate of cells and subcellular fractions were prepared as described previously (Chadani et al., 2010) with minor modifications. Briefly, CH131 harbouring appropriate plasmid was grown in LB. At the mid-log phase, IPTG was added to the final concentration of 0.5 mm, and the cells were incubated for 1.5 h to induce protein expression. Cells were harvested, washed with ice-cold STE (0.1 M NaCl, 10 mm Tris- HCl ph 8.0, 1 mm EDTA), and then suspended in TMN buffer (25 mm Tris-HCl ph 7.5, 10 mm MgCl 2, 100 mm NH 4Cl, 7 mm 2-mercaptoethanol, 0.2 mm PMSF; 1 ml for 100 mg cells). Lysozyme was added to the final concentration of 250 mg ml -1 and, the 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), and the supernatant was recovered as a crude lysate, which was fractionated into S30 and P30 fractions by ultracentrifugation (25 krpm, 30 min, 4 C using Beckman Optima-TM TLM Ultracentrifuge and TLA100.3). The S30 fraction was further fractionated into precipitate and supernatant fractions by ultracentrifugation (50 krpm, 100 min, 4 C). The supernatant was the S100 fraction. The precipitates were rinsed with TMN buffer, suspended in TMN buffer whose volume was equal to that of the supernatant, and centrifuged again at g to remove insoluble components. The resulting supernatant was recovered as the P100 fraction. Sucrose gradient fractionation of in vitro translation sample was performed as described previously (Chadani et al., 2010) with some exceptions. One hundred and fifty microlitres of sample treated with 200 mg ml -1 of chloramphenicol was applied onto a 10 40% sucrose gradient containing 25 mm Tris-HCl ph 7.4, 100 mm NH 4Cl, 10 mm MgCl 2 and 1 mm DTT, prepared in Thinwall Polyallomer Centrifuge Tubes (13 51 mm, Beckman) and fractionated by ultracentrifugation (Beckman Optima-TM L-80, SW50.1- Ti, 33 krpm, 2.7 h, 4 C). After centrifugation, each fraction (approximately 100 ml) was collected. Cell-free translation The S30 extracts of CH101 and CH161 were prepared as described previously (Chadani et al., 2010). Translation of in vitro prepared non-stop mrna was performed in the mixture containing 62.5 mm HEPES-KOH ph 7.4, 1.7 mm DTT, 174 mg ml -1 E. coli trna, 50 mm creatine phosphate, 2% PEG 8000, 20 mg ml -1 folinic acid, 1 mm each amino acid, 1.25 mm ATP, 0.8 mm GTP, 0.63 mm cyclic AMP, 30 mm ammonium acetate, 175 mm potassium glutamate, 1.5 mm spermidine, 80 mg ml -1 creatine kinase, 100 mg ml -1 mrna, 2.5 mm magnesium acetate, 50 ng ml -1 anti-ssra oligonucleotide (5 -TTAAGCTGCTAAAGCGTAGTTTTCGTCG TTTGCGACTA-3 ; Hanes and Plückthun, 1997; Chadani et al., 2010), and 30% volume of S30 extract for 30 min at 37 C. After the translation reaction, 100 nm of purified protein components or 10 mm of puromycin was added, and incubation was continued for another 10 min. Then, translation was stopped by adding equal volume of SDS sample buffer (100 mm Tris-HCl ph 6.8, 10% 2-mercaptoethanol, 4% SDS, 0.02% bromophenol blue and 20% glycerol). Non-stop mrna used as a template for cell-free translation was prepared by transcribing HindIII-digested pch353 using ScriptMAX Thermo T7 Transcription Kit (TOYOBO). The transcript, which contains crp ORF truncated at the 250th codon, was then purified using MicroSpin G-50 column (GE healthcare). CRP-ST mrna was prepared in the same way except that HindIII-digested pch343 was used as a template. His 6-tagged proteins used in the cell-free reaction system were purified from the extract of BL21/pCH201 (His 6-ArfA), BL21/pCH221 (His 6-ArfA-A18T), BL21/pCH400 (His 6-YaeJ) or BL21/pCH410 (His 6-YaeJ-G27A) cells, which had been grown for 3 h in the presence of 0.5 mm IPTG using Ni-NTA agarose (Qiagen), according to the supplier s instruction. Bis-Tris SDS-PAGE analysis After addition of SDS sample buffer, samples were incubated for 2 h at 37 C and analysed by Western blotting after 12% Bis-Tris NuPAGE (Invitrogen), of which the running buffer system contained MOPS.