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• W2084164517 abstract "The role of ribosome-binding molecular chaperones in protein folding is not yet well understood. Trigger factor (TF) is the first chaperone to interact with nascent polypeptides as they emerge from the bacterial ribosome. It binds to the ribosome as a monomer but forms dimers in free solution. Based on recent crystal structures, TF has an elongated shape, with the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and the N-terminal ribosome binding domain positioned at opposite ends of the molecule and the C-terminal domain, which forms two arms, positioned in between. By using site specifically labeled TF proteins, we have demonstrated that all three domains of TF interact with nascent chains during translation. Interactions with the PPIase domain were length-dependent but independent of PPIase activity. Interestingly, with free TF, these same sites were found to be involved in forming the dimer interface, suggesting that dimerization partially occludes TF-nascent chain binding sites. Our data indicate the existence of two regions on TF along which nascent chains can interact, the NC-domains as the main site and the PPIase domain as an auxiliary site. The role of ribosome-binding molecular chaperones in protein folding is not yet well understood. Trigger factor (TF) is the first chaperone to interact with nascent polypeptides as they emerge from the bacterial ribosome. It binds to the ribosome as a monomer but forms dimers in free solution. Based on recent crystal structures, TF has an elongated shape, with the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and the N-terminal ribosome binding domain positioned at opposite ends of the molecule and the C-terminal domain, which forms two arms, positioned in between. By using site specifically labeled TF proteins, we have demonstrated that all three domains of TF interact with nascent chains during translation. Interactions with the PPIase domain were length-dependent but independent of PPIase activity. Interestingly, with free TF, these same sites were found to be involved in forming the dimer interface, suggesting that dimerization partially occludes TF-nascent chain binding sites. Our data indicate the existence of two regions on TF along which nascent chains can interact, the NC-domains as the main site and the PPIase domain as an auxiliary site. Trigger factor (TF) 3The abbreviations used are: TF, trigger factor; IPTG, isopropyl 1-thio-β-d-galactopyranoside; Kan, kanamycin; Tet, tetracycline; pBpa, para-benzoyl-l-phenylalanine.3The abbreviations used are: TF, trigger factor; IPTG, isopropyl 1-thio-β-d-galactopyranoside; Kan, kanamycin; Tet, tetracycline; pBpa, para-benzoyl-l-phenylalanine. was first identified based on its ability to maintain the precursor of the outer membrane protein A pro-OmpA in a non-aggregated form, competent for translocation across the Escherichia coli inner membrane in vitro (1Crooke E. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5216-5220Crossref PubMed Scopus (165) Google Scholar). Subsequent studies have demonstrated that TF has a general role in cytosolic protein folding that overlaps partially with that of the Hsp70 chaperone system, DnaK, DnaJ, and GrpE (2Deuerling E. Schulze-Specking A. Tomoyasu T. Mogk A. Bukau B. Nature. 1999; 400: 693-696Crossref PubMed Scopus (401) Google Scholar, 3Teter S.A. Houry W.A. Ang D. Tradler T. Rockabrand D. Fischer G. Blum P. Georgopoulos C. Hartl F.U. Cell. 1999; 97: 755-765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Although the tig and dnaK genes are not individually essential, their combined deletion at temperatures >30 °C results in synthetic lethality, apparently because of large scale protein aggregation (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar). However, the combined deletion of both systems is possible at lower temperature, and the resulting ΔtigΔdnaK cells can be adapted to growth at up to 30 °C (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar, 5Vorderwulbecke S. Kramer G. Merz F. Kurz T.A. Rauch T. Zachmann-Brand B. Bukau B. Deuerling E. FEBS Lett. 2004; 559: 181-187Crossref PubMed Scopus (92) Google Scholar). Other chaperones, such as SecB and GroEL may partially compensate for the loss of TF and DnaK under these conditions (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar, 5Vorderwulbecke S. Kramer G. Merz F. Kurz T.A. Rauch T. Zachmann-Brand B. Bukau B. Deuerling E. FEBS Lett. 2004; 559: 181-187Crossref PubMed Scopus (92) Google Scholar). The absence of TF causes the flux of newly synthesized polypeptides through DnaK to increase (3Teter S.A. Houry W.A. Ang D. Tradler T. Rockabrand D. Fischer G. Blum P. Georgopoulos C. Hartl F.U. Cell. 1999; 97: 755-765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). TF is a ribosome-associated protein (6Blaha G. Wilson D.N. Stoller G. Fischer G. Willumeit R. Nierhaus K.H. J. Mol. Biol. 2003; 326: 887-897Crossref PubMed Scopus (43) Google Scholar, 7Hesterkamp T. Deuerling E. Bukau B. J. Biol. Chem. 1997; 272: 21865-21871Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Maier R. Eckert B. Scholz C. Lilie H. Schmid F.X. J. Mol. Biol. 2003; 326: 585-592Crossref PubMed Scopus (76) Google Scholar) that has been shown to increase the folding efficiency of certain multidomain proteins concomitant with delaying their folding relative to translation (9Agashe V.R. Guha S. Chang H.C. Genevaux P. Hayer-Hartl M. Stemp M. Georgopoulos C. Hartl F.U. Barral J.M. Cell. 2004; 117: 199-209Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Ribosome binding is mediated by the N-terminal 118 residues of TF (7Hesterkamp T. Deuerling E. Bukau B. J. Biol. Chem. 1997; 272: 21865-21871Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), specifically by a loop region consisting of amino acids Phe-44, Arg-45, and Lys-46 that contacts the L23 protein in the 50 S ribosomal subunit (10Kramer G. Rauch T. Rist W. Vorderwulbecke S. Patzelt H. Schulze-Specking A. Ban N. Deuerling E. Bukau B. Nature. 2002; 419: 171-174Crossref PubMed Scopus (269) Google Scholar). Recently, the crystal structure of full-length TF and co-crystal structures of the archaeal and the eubacterial 50 S ribosomal subunit with the N-domain of TF were reported (11Schlunzen F. Wilson D.N. Tian P. Harms J.M. McInnes S.J. Hansen H.A. Albrecht R. Buerger J. Wilbanks S.M. Fucini P. Structure (Camb.). 2005; 13: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (301) Google Scholar, 13Baram D. Pyetan E. Sittner A. Auerbach-Nevo T. Bashan A. Yonath A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12017-12022Crossref PubMed Scopus (91) Google Scholar, 14Ludlam A.V. Moore B.A. Xu Z. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13436-13441Crossref PubMed Scopus (57) Google Scholar). In these structures, the TF N-domain interacts with proteins L23, L29, and the 23 S rRNA near the peptide exit tunnel. Upon binding to eubacterial ribosomes, a conformational change in the structure of the N-domain was observed (13Baram D. Pyetan E. Sittner A. Auerbach-Nevo T. Bashan A. Yonath A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12017-12022Crossref PubMed Scopus (91) Google Scholar), resulting in increased solvent exposure of hydrophobic surface area compared with the structure of free TF (11Schlunzen F. Wilson D.N. Tian P. Harms J.M. McInnes S.J. Hansen H.A. Albrecht R. Buerger J. Wilbanks S.M. Fucini P. Structure (Camb.). 2005; 13: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 13Baram D. Pyetan E. Sittner A. Auerbach-Nevo T. Bashan A. Yonath A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12017-12022Crossref PubMed Scopus (91) Google Scholar). Consistent with such a conformational change, a recent study has demonstrated that fluorescently labeled full-length TF undergoes a structural expansion upon ribosome binding. The rates with which TF relaxed to its compact conformation upon ribosome departure varied depending on the characteristics of the nascent chain (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). A marked delay in the relaxation rate was correlated with the presence of nascent chain segments with high mean hydrophobicity. The model protein firefly luciferase was shown to have two such preferred TF binding sites (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). Previous analysis of the primary structure of TF identified three distinct domains (10Kramer G. Rauch T. Rist W. Vorderwulbecke S. Patzelt H. Schulze-Specking A. Ban N. Deuerling E. Bukau B. Nature. 2002; 419: 171-174Crossref PubMed Scopus (269) Google Scholar, 12Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (301) Google Scholar, 16Kristensen O. Gajhede M. Structure (Camb.). 2003; 11: 1547-1556Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In the recent crystal structures, TF displays an elongated shape (12Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (301) Google Scholar, 14Ludlam A.V. Moore B.A. Xu Z. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13436-13441Crossref PubMed Scopus (57) Google Scholar) in which the PPIase domain (residues 150-247) is connected with the N-domain (residues 1-150) via a long extension, so that it is positioned at the other end of the molecule, whereas the C-domain lies between the N-domain and the PPIase domain (Fig. 1A). The function of the PPIase domain remains unclear. Although prolyl-cis/trans-isomerase activity has been detected in vitro, the domain is dispensable for TF function in vivo (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar, 17Kramer G. Rutkowska A. Wegrzyn R.D. Patzelt H. Kurz T.A. Merz F. Rauch T. Vorderwulbecke S. Deuerling E. Bukau B. J. Bacteriol. 2004; 186: 3777-3784Crossref PubMed Scopus (72) Google Scholar). Moreover, the PPIase domain binds preferentially to peptide segments of at least eight amino acids that are enriched in basic and aromatic amino acids but do not necessarily contain proline residues, suggesting a more general chaperone function (18Patzelt H. Rudiger S. Brehmer D. Kramer G. Vorderwulbecke S. Schaffitzel E. Waitz A. Hesterkamp T. Dong L. Schneider-Mergener J. Bukau B. Deuerling E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14244-14249Crossref PubMed Scopus (148) Google Scholar, 19Kramer G. Patzelt H. Rauch T. Kurz T.A. Vorderwulbecke S. Bukau B. Deuerling E. J. Biol. Chem. 2004; 279: 14165-14170Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The C-domain shows structural similarity to the periplasmic chaperone SurA (20Maier T. Ferbitz L. Deuerling E. Ban N. Curr. Opin. Struct. Biol. 2005; 15: 204-212Crossref PubMed Scopus (78) Google Scholar) and to MPN555 of Mycoplasma pneumoniae, a predicted protein of unknown function (21Schulze-Gahmen U. Aono S. Chen S. Yokota H. Kim R. Kim S.H. Acta. Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 1343-1347Crossref PubMed Scopus (12) Google Scholar). This domain can be divided into two subdomains consisting of arm 1 and arm 2. The TF structure (12Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (301) Google Scholar) suggests that these arms and a segment of the N-domain constitute a crevice that may interact with polypeptides emerging from the exit tunnel (22Merz F. Hoffmann A. Rutkowska A. Zachmann-Brand B. Bukau B. Deuerling E. J. Biol. Chem. 2006; 281: 31963-31971Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Numerous ribosome-associated polypeptide chains, including membrane proteins and cytosolic proteins, have been chemically or site-specifically cross-linked to TF (22Merz F. Hoffmann A. Rutkowska A. Zachmann-Brand B. Bukau B. Deuerling E. J. Biol. Chem. 2006; 281: 31963-31971Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Ullers R.S. Luirink J. Harms N. Schwager F. Georgopoulos C. Genevaux P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7583-7588Crossref PubMed Scopus (95) Google Scholar, 25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar, 26Hoffmann A. Merz F. Rutkowska A. Zachmann-Brand B. Deuerling E. Bukau B. J. Biol. Chem. 2006; 281: 6539-6545Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 27Houben E.N. Zarivach R. Oudega B. Luirink J. J. Cell Biol. 2005; 170: 27-35Crossref PubMed Scopus (47) Google Scholar, 28Eisner G. Koch H.G. Beck K. Brunner J. Muller M. J. Cell Biol. 2003; 163: 35-44Crossref PubMed Scopus (56) Google Scholar). These interactions are abolished with a ribosome binding-deficient mutant TF F44A/R45A/K46A and thus depend on the ability of TF to dock onto the ribosome in close proximity to the opening of the polypeptide exit tunnel (25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar). TF displays a 30-fold increase in affinity for translating versus non-translating ribosomes (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar, 29Hesterkamp T. Hauser S. Lutcke H. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4437-4441Crossref PubMed Scopus (203) Google Scholar, 30Raine A. Lovmar M. Wikberg J. Ehrenberg M. J. Biol. Chem. 2006; 281: 28033-28038Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), which may be mediated by the exposure of hydrophobic sequence motifs in elongating nascent chains (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). To determine which domain(s) of TF interact with elongating nascent polypeptides, we placed photoreactive probes at specific sites in each domain of TF and added the purified probe containing proteins to a reconstituted in vitro translation system essentially free of chaperones (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar, 31Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1268) Google Scholar). We have demonstrated here that probes in the N- and C-domains lining the proposed binding crevice are adjacent to the nascent chain. The PPIase domain also participates in nascent chain interaction in a manner dependent on chain length and the specific polypeptide analyzed. Probe sites in the N- and C-domains that cross-linked to nascent chains (but not sites in the PPIase domain) proved to be involved in forming the TF dimer interface for free TF in solution. Plasmids—The pYC-JYCUA and pBKpBpa plasmids were a generous gift from laboratory of Dr. Peter Schultz (32Chin J.W. Schultz P.G. Chembiochem. 2002; 3: 1135-1137Crossref PubMed Scopus (116) Google Scholar). The wild-type TF or the triple mutant F44A/R45A/K46A genes were subcloned into the pYC-JYCUA vector, resulting in the addition of C-terminal Myc and His6 tags. This vector also encoded the mutRNATYRCUA under the control of the constitutively active lpp promoter, the terminator rrnC as well as Tetr. These vectors were used as templates for site-directed mutagenesis in which TF codons encoding residues 14, 34, 73, 88, 118, 168, 177, 185, 198, 233, 320, 322, 373, 377, 378, 384, 387, or 419 were changed to encode an amber stop codon (33Etchells S.A. Meyer A.S. Yam A.Y. Roobol A. Miao Y. Shao Y. Carden M.J. Skach W.R. Frydman J. Johnson A.E. J. Biol. Chem. 2005; 280: 28118-28126Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The firefly luciferase (luc) gene was in plasmid pET15d as was the Luc5amb gene. α-Synuclein was in plasmid pSTS500, as described previously (25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar). tig was also subcloned into the pOFX vector under an IPTG-inducible promoter (pOFX-TF). The pBKpBpa plasmid encoded the Methanococcus jannaschii tyrosyl tRNA synthetase under the glnRS promoter (32Chin J.W. Schultz P.G. Chembiochem. 2002; 3: 1135-1137Crossref PubMed Scopus (116) Google Scholar). Protein Expression and Purification—BL21 cells were co-transformed with plasmids pBKpBpa and pYC-JYCUA (encoding one of the TF mutants described above). Cells were grown in LB medium supplemented with 30 μg/ml kanamycin (Kan), 25 μg/ml tetracycline (Tet), and 1 mm para-benzoyl-l-phenylalanine (pBpa) to an A600 = 0.6 at 30 °C. Protein expression was induced with 0.2% d-arabinose for 4 h. His6-tagged TF proteins were isolated from the soluble fraction of cell extracts on Ni2+-nitrilotriacetic acid-agarose (Qiagen) followed by anion exchange chromatography on a Resource Q column (Amersham Biosciences). Proteins were stored at -80 °C in amber-colored tubes. TF and TF-NC (a version composed of the N- and C-domains only), which each had a cleavable N-terminal His6 tag, were purified according to Ref. (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). The linker sequence between the N- and C-domains was GTSAAAG (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). In Vivo Activity Test of TF and TF Variants—E. coli MG1655ΔdnaK::CmrΔtig cells 4P. Genevaux, unpublished data. were co-transformed as above and grown at 23 °C in LB agar supplemented with 30 μg/ml Kan and 25 μg/ml Tet. After 36 h, colonies were inoculated in LB medium supplemented with Kan/Tet, as above, and grown at 23 °C overnight. The growth of cells serially diluted on LB Kan/Tet plates containing either 0.2% arabinose or 1 mm pBpa or both and incubated at 23, 30, and 37 °C was examined. Only the TF constructs that rescued growth at 37 °C were used in subsequent cross-linking experiments. The F44A/R45A/K46A construct, defective in ribosome binding, did not support growth at 37 °C and served as a negative control in subsequent cross-linking experiments. In Vitro Cross-linking Reactions with TF and TF Single Site Mutants—In vitro runoff transcription/translation of Luc constructs lacking stop codons was performed in the PURE SYSTEM (Post Genome Institute Co. Ltd., Tokyo, Japan) (31Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1268) Google Scholar) at 30 °C for 55 min in the presence of 0.8 μCi/μl[35S]Met. When indicated, TF variants labeled with pBpa were added to a final concentration of 1 μm. Reactions were stopped by the addition of 0.15 μl of chloramphenicol (34 mg/ml) and incubated for 10 min on ice. Next, the samples were placed under a 500 W Mercury Arc lamp (LOT-Oriel, Darmstadt, Germany) for 2-60 min on ice. A 20-μl aliquot of a 25-μl reaction was layered over a 100-μl sucrose cushion (0.5 m sucrose, 15 mm MgCl2, 100 mm KOAc, and 200 mm Hepes, pH 7.5) and centrifuged in a TLA 100 rotor (Beckman) at 100,000 × g for 20 min at 4 °C. Pelleted ribosome-nascent chain complexes were resuspended in 100 μl of water, and then RNase A (protease-free) (Roche Applied Science) and EDTA were added to final concentrations of 100 μg/ml and 10 mm, respectively, followed by incubation at 37 °C for 10 min. After trichloroacetic acid precipitation, the reactions were resolved on 4-10% SDS-PAGE followed by autoradiography. The identities of the photoadducts were confirmed by immunoprecipitation with anti-TF antibodies. Western Blots of TF and TF Single Site Mutants—5 μm purified TF and TF single site mutants were photolyzed for 15 min, loaded on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Whatman). The membranes were blocked with 5% (w/v) skim milk powder in TBST (20 mm Tris-HCl, pH 7.6, 136 mm NaCl, and 0.1% (w/v) Tween 20), washed three times with TBST, and then a primary antibody against the Myc tag (American Type Culture Collection) was used to probe the membrane at a dilution of 1:5000 followed by three washes and detection with a secondary anti-rabbit horseradish peroxidase conjugate (Sigma) used at 1:5000 in TBST. The signal was developed using the ECL Plus Western blotting detection system (Amersham Biosciences) and visualized using Imagereader LAS-3000 (Fuji). In Vitro Proteinase K Assay—All proteinase K assays were performed as described previously (25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar). In brief, in vitro translations of Luc-nascent chains of three lengths (92, 125, and 280 residues) were preformed either in the absence of chaperone or in the presence of 1 μm TF or 1 μm TF-NC. Each reaction was stopped by the addition of chloramphenicol, and then samples were treated for the indicated time with proteinase K (Merck). The reactions were then trichloroacetic acid-precipitated and separated by gel electrophoresis, and the resulting radioactively labeled p-tRNA-Luc-nascent chains were quantified using an Imagereader LAS-3000 (Fuji). In Vitro Cross-linking Reaction with the Probe in the Nascent Chain—In vitro runoff transcription/translation reactions were preformed with Luc-nascent chains of various lengths from 77 to 280 amino acids (as above). Nascent chains contained a single amber stop codon at position 5 from the N terminus, as previously described (25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar). TF or TF-NC was present at 1 μm. Characterization of TF with Photoreactive Probes at Different Locations—To determine which regions of TF interact with the nascent chain as it emerges from the ribosome, we made use of a recently developed technology (32Chin J.W. Schultz P.G. Chembiochem. 2002; 3: 1135-1137Crossref PubMed Scopus (116) Google Scholar) allowing for in vivo expression of a protein with a benzophenone photoreactive probe incorporated at a specific site through amber codon suppression. Benzophenone, a phenylalanine analog, consists of two phenol rings with the photoreactive moiety residing in between the two rings (supplemental Fig. 1). Purified TF proteins with single probes incorporated were then added to a fully reconstituted bacterial translation system lacking chaperones. Following runoff translation in the presence of TF and upon exposure to UV light, covalent bonds formed between TF and nascent chains adjacent to the photoreactive probe in TF. Several sites in each domain of TF (Fig. 1A and supplemental Fig. 2A), including residues 34, 88, 118, 185, 233, 320, 373, and 419, were chosen for probe incorporation. We confirmed that the amber codon was suppressed and the TF variants were translated, resulting in full-length proteins with C-terminal Myc and His6 tags (Fig. 1B). The benzophenone-carrying proteins were expressed in MG1655 cells from an arabinose promoter at a level ∼2-fold above that of endogenous TF (data not shown). In contrast, under the same conditions, recombinant wild-type TF was expressed at substantially higher levels, because it does not contain an amber site and does not rely on suppression for completion of translation. Truncated TF products that were not suppressed and therefore did not contain probes or C-terminal tags were not observed after purification. To test whether the probe-containing TF proteins were functional, we used an in vivo complementation assay. ΔdnaKΔtig cells are not viable at 37 °C unless TF is expressed from a plasmid (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar, 17Kramer G. Rutkowska A. Wegrzyn R.D. Patzelt H. Kurz T.A. Merz F. Rauch T. Vorderwulbecke S. Deuerling E. Bukau B. J. Bacteriol. 2004; 186: 3777-3784Crossref PubMed Scopus (72) Google Scholar). TF14, TF34, TF73, TF88, TF118, TF185, TF233, TF320, TF373, and TF419, carrying the probe at the indicated residue, rescued growth at 37 °C (Fig. 1C and supplemental Fig. 2C). Because overexpression of wild-type TF in MG1655 ΔdnaKΔtig cells is toxic (4Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (141) Google Scholar), an IPTG-inducible promoter was used to express wild-type TF at lower levels. However, when probes were placed at positions 377, 378, or 387 in arm 2 of TF, no E. coli growth was observed. Because the mutant proteins were produced (data not shown), incorporation of the probe at these positions likely interferes with TF chaperone activity or folding of the protein. These sites were therefore not further analyzed. Notably, probe incorporation at positions 233 and 185 in the PPIase domain resulted in a partial loss of PPIase activity measured with RNase T1 as a substrate, consistent with previous mutational studies (34Tradler T. Stoller G. Rucknagel K.P. Schierhorn A. Rahfeld J.U. Fischer G. FEBS Lett. 1997; 407: 184-190Crossref PubMed Scopus (60) Google Scholar) (and data not shown). Tryptophan fluorescence and circular dichroism measurements demonstrated the absence of significant conformational differences in the probe-containing mutant proteins compared with wild-type TF (supplemental Fig. 3 and supplemental Table 1). Photocross-linking of TF Single Site Mutants to Ribosome-nascent Chain Complexes—Using the TF single site mutants with probes in each of its domains, in vitro translation reactions were performed to determine which probes were adjacent to the nascent chain as it emerged from the ribosome. Luc was chosen as a model substrate, because it efficiently interacts with TF upon translation (9Agashe V.R. Guha S. Chang H.C. Genevaux P. Hayer-Hartl M. Stemp M. Georgopoulos C. Hartl F.U. Barral J.M. Cell. 2004; 117: 199-209Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar, 25Tomic S. Johnson A.E. Hartl F.U. Etchells S.A. FEBS Lett. 2006; 580: 72-76Crossref PubMed Scopus (32) Google Scholar). Luc-nascent chains of 60, 77, and 164 amino acids in length were stalled on the ribosome and analyzed. Cross-links to each domain of TF were observed for the 60- and 164-mer (Fig. 2, A and C). N-domain interactions were observed with TF34 to Luc-nascent chains of 60, 77, and 164 amino acids in length (Fig. 2). TF320 and TF373, in which probes are in the tip of arm 1 and arm 2 of the C-domain, respectively, cross-linked to all lengths of Luc-nascent chains tested (Fig. 2). Photoadducts with TF320 were the most intense and hence were used as a reference in all panels of Fig. 2. Interestingly, TF320 resulted in two photoadducts to the Luc 60- and 77-mers differing in size, presumably because of TF binding to two independent sites on the nascent chain (Fig. 2, A and B). Importantly, no cross-links were observed when the probes were placed on the back surface of the TF molecule at positions 419 (Fig. 2) or 118 (supplemental Fig. 2D), thus suggesting that the nascent chain moves through or spans the proposed binding crevice accessible from the front side of the molecule. No cross-links were observed with the TF320 version of the F44A/R45A/K46A mutant, confirming that ribosome binding is a prerequisite for nascent chain interaction (Fig. 2). Cross-linking to the PPIase domain displayed a nascent chain length dependence. Short nascent chains of 60 amino acids in length cross-linked to positions 185 and 233 in the PPIase domain, whereas nascent chains of 77 amino acids did not (Fig. 2) (15Kaiser C.M. Chang H.-C. Agashe V.R. Laksmipathy S.K. Etchells S.A. Hartl F.U. Barral J.M. Nature. 2006; 444: 455-460Crossref PubMed Scopus (182) Google Scholar). Thus, between the lengths of 60 and 77 amino acids, a portion of the Luc-nascent chain changes position relative to the probes in the PPIase domain. Taking the dimensions of TF in the crystal structure into account, cross-linking of the short 60-mer chain to the PPIase domain suggests that the nascent polypeptide must be in a rather extended conformation. Interestingly, longer nascent chains of 164 amino acids again cross-linked to TF233 but not to TF185 (even after 30 min of irradiation; data not shown), suggesting that different portions of the Luc nascent chain are adjacent to the probe at position 233 as the chain elongates from 60 to 164 amino acids (Fig. 2C). Luc 125-mers were also shown to cross-link to TF233 (Fig. 3B). The differential cross-linking to TF233 and/or TF185 suggests that the interaction with the PPIase domain does not merely result from collision with a flexible nascent chain but rather reflects a more specific participation of this domain in nascent chain binding. The yield of Luc folding is known to be improved by t" @default.
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• W2084164517 date "2007-04-01" @default.
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• W2084164517 title "Identification of Nascent Chain Interaction Sites on Trigger Factor" @default.
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