Crystal Structure of the Mor Protein of Bacteriophage Mu, a Member of the Mor/C Family of Transcription Activators
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m313555200
ISSN1083-351X
AutoresMuthiah Kumaraswami, Martha M. Howe, Hee-Won Park,
Tópico(s)RNA and protein synthesis mechanisms
ResumoTranscription from the middle promoter, Pm, of bacteriophage Mu requires the phage-encoded activator protein Mor and bacterial RNA polymerase. Mor is a sequence-specific DNA-binding protein that mediates transcription activation through its interactions with the C-terminal domains of the α and σ subunits of bacterial RNA polymerase. Here we present the first structure for a member of the Mor/C family of transcription activators, the crystal structure of Mor to 2.2-Å resolution. Each monomer of the Mor dimer is composed of two domains, the N-terminal dimerization domain and C-terminal DNA-binding domain, which are connected by a linker containing a β strand. The N-terminal dimerization domain has an unusual mode of dimerization; helices α1 and α2 of both monomers are intertwined to form a four-helix bundle, generating a hydrophobic core that is further stabilized by antiparallel interactions between the two β strands. Mutational analysis of key leucine residues in helix α1 demonstrated a role for this hydrophobic core in protein solubility and function. The C-terminal domain has a classical helix-turn-helix DNA-binding motif that is located at opposite ends of the elongated dimer. Since the distance between the two helix-turn-helix motifs is too great to allow binding to two adjacent major grooves of the 16-bp Mor-binding site, we propose that conformational changes in the protein and DNA will be required for Mor to interact with the DNA. The highly conserved glycines flanking the β strand may act as pivot points, facilitating the conformational changes of Mor, and the DNA may be bent. Transcription from the middle promoter, Pm, of bacteriophage Mu requires the phage-encoded activator protein Mor and bacterial RNA polymerase. Mor is a sequence-specific DNA-binding protein that mediates transcription activation through its interactions with the C-terminal domains of the α and σ subunits of bacterial RNA polymerase. Here we present the first structure for a member of the Mor/C family of transcription activators, the crystal structure of Mor to 2.2-Å resolution. Each monomer of the Mor dimer is composed of two domains, the N-terminal dimerization domain and C-terminal DNA-binding domain, which are connected by a linker containing a β strand. The N-terminal dimerization domain has an unusual mode of dimerization; helices α1 and α2 of both monomers are intertwined to form a four-helix bundle, generating a hydrophobic core that is further stabilized by antiparallel interactions between the two β strands. Mutational analysis of key leucine residues in helix α1 demonstrated a role for this hydrophobic core in protein solubility and function. The C-terminal domain has a classical helix-turn-helix DNA-binding motif that is located at opposite ends of the elongated dimer. Since the distance between the two helix-turn-helix motifs is too great to allow binding to two adjacent major grooves of the 16-bp Mor-binding site, we propose that conformational changes in the protein and DNA will be required for Mor to interact with the DNA. The highly conserved glycines flanking the β strand may act as pivot points, facilitating the conformational changes of Mor, and the DNA may be bent. Transcription initiation is a major control point for gene expression in prokaryotes (1Reznikoff W.S. Siegele D.A. Cowing D.W. Gross C.A. Annu. Rev. Genet. 1985; 19: 355-387Crossref PubMed Scopus (123) Google Scholar) and is regulated by a number of gene- and regulon-specific proteins. In many cases this regulation is mediated by protein-protein interactions between transcriptional activators or repressors and one or more subunits of the bacterial DNA-dependent RNA polymerase (2Hochschild A. Dove S.L. Cell. 1998; 92: 597-600Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Bacteriophage Mu is a temperate phage that infects several species of enteric bacteria, including Escherichia coli K-12 (3Symonds N.A. Toussaint A. van de Putte P. Howe M.M. Phage Mu. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1987Google Scholar, 4Howe M.M. Busby S.J.W. Thomas C.M. Brown N.L. Molecular Microbiology. Springer-Verlag, Heidelberg1997: 65-80Google Scholar). The lytic cycle is characterized by a regulatory cascade with three phases of gene expression: early, middle, and late (5Marrs C.F. Howe M.M. Virology. 1990; 174: 192-203Crossref PubMed Scopus (22) Google Scholar) (Fig. 1A). The early promoter, Pe, has typical -10 and -35 sequences and is recognized directly by the bacterial RNA polymerase (16Krause H.M. Higgins N.P. J. Biol. Chem. 1986; 261: 3744-3752Abstract Full Text PDF PubMed Google Scholar). The middle and late promoters have recognizable -10 hexamers but lack the -35 hexamer; transcription from these promoters requires the phage-encoded proteins Mor 1The abbreviations used are: Mor, middle operon regulator protein; TrpR, tryptophan operon repressor; HTH, helix-turn-helix DNA-binding motif; r.m.s.d., root mean square deviation; α-CTD, C-terminal domain of α subunit of bacterial RNA polymerase; σ-CTD, C-terminal domain of σ subunit of bacterial RNA polymerase; TEMED, tetramethylethylenediamine; IPTG, isopropyl-β-d-thiogalactopyranoside; Cm, chloramphenicol; Ap, ampicillin. and C, respectively (17Margolin W. Rao G. Howe M.M. J. Bacteriol. 1989; 171: 2003-2018Crossref PubMed Google Scholar, 18Stoddard S.F. Howe M.M. J. 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Because there is no amino acid sequence homology between Mor and previously studied transcription factors, Mor and C define a new family of transcription factors that we call the Mor/C family. Mor binds to the middle promoter Pm as a homodimer, recognizing a 16-bp region from -36 to -51 with respect to the transcription start site at +1 (25Artsimovitch I. Howe M.M. Nucleic Acids Res. 1996; 24: 450-457Crossref PubMed Google Scholar). Mutational analysis of the middle promoter sequence (25Artsimovitch I. Howe M.M. Nucleic Acids Res. 1996; 24: 450-457Crossref PubMed Google Scholar) identified an imperfect dyadsymmetry element within the Mor-binding site (Fig. 1B). It also revealed a bias between the two halves of the Morbinding site, showing a greater importance of the down-stream half-site for Mor binding and promoter activity (25Artsimovitch I. Howe M.M. Nucleic Acids Res. 1996; 24: 450-457Crossref PubMed Google Scholar). Transcription activation of the middle promoter by Mor requires the C-terminal domains of both the α (α-CTD) and σ (σ-CTD) subunits of bacterial RNA polymerase (26Artsimovitch I. Murakami K. Ishihama A. Howe M.M. J. Biol. Chem. 1996; 271: 32343-32348Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Binding of Mor and RNA polymerase to the promoter introduces a strand separation or distortion involving promoter positions -32 to -34 in addition to positions -12 to -1 (27Artsimovitch I. Kahmeyer-Gabbe M. Howe M.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9408-9413Crossref PubMed Scopus (10) Google Scholar). Based on these results, it has been proposed that the mechanism for Mor-dependent middle promoter activation involves Mormediated recruitment of RNA polymerase to the promoter and/or isomerization of the closed complex to an open complex through its interaction with the α-CTD and σ-CTD of RNA polymerase (27Artsimovitch I. Kahmeyer-Gabbe M. Howe M.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9408-9413Crossref PubMed Scopus (10) Google Scholar) (Fig. 1C). In an effort to gain a better understanding of Mor function, we determined the crystal structure of Mor to a resolution of 2.2 Å. The structure reveals an unusual mode of dimerization along with a classical helix-turn-helix DNA-binding motif. Most interesting, in the structure, the HTH motifs of Mor are located too far apart to interact with the two adjacent major grooves of DNA; thus, conformational changes in Mor may be needed for it to bind to DNA. Because Mor does not share sequence or structural similarities to other characterized proteins, the Mor structure could very well serve as a paradigm for the Mor/C family of proteins. Media, Chemicals, Enzymes, Columns, and Analytical Services— Routine cell growth and protein overexpression were done in LB medium (28Howe M.M. Virology. 1973; 54: 93-101Crossref PubMed Scopus (123) Google Scholar), whereas cultures for β-galactosidase assays were grown in minimal medium with casamino acids (M9CA) (17Margolin W. Rao G. Howe M.M. J. Bacteriol. 1989; 171: 2003-2018Crossref PubMed Google Scholar). When needed, medium was supplemented with chloramphenicol (Cm) at 25 μg/ml and ampicillin (Ap) at 40 μg/ml. MacConkey lactose plates with 50 g/liter of MacConkey agar (Difco) was used for plate phenotyping. Chloramphenicol was purchased from Sigma, and ampicillin was obtained from U. S. Biochemical Corp. Isopropyl-β-d-thiogalactopyranoside (IPTG) and o-nitrophenyl-β-galactopyranoside were from American Bioorganics. Acrylamide, bisacrylamide, TEMED, SDS, ammonium persulfate, and Biospin columns were purchased from Bio-Rad. Chloroform and glycerol were obtained from Fisher, and thiomersal and K2PtCl4 were from Aldrich. Imidazole was purchased from Sigma, and guanidine hydrochloride was from Hampton Research. Seakem- and Nusieve (both genome technology grade)-agarose were from FMC Bioproducts. Talon spin columns used for small scale protein purification were from Clontech, and the nickel-nitrilotriacetic acid-agarose resin for large scale preparations was from Qiagen. The Superdex-75 gel filtration column was from Amersham Biosciences. The restriction enzymes HindIII and PstI were from New England Biolabs. The Thermus aquaticus polymerase and T4 DNA ligase were from Roche Applied Science; T4 polynucleotide kinase was obtained from Promega Corp. Shrimp alkaline phosphatase was purchased from U. S. Biochemical Corp. Oligonucleotides were purchased from Integrated DNA Technologies. Automated DNA sequencing was performed by the Molecular Resource Center of the University of Tennessee Health Science Center. Mass spectroscopy and N-terminal protein sequencing were performed by the Hartwell Center for Biotechnology and Bioinformatics of St. Jude Children's Research Hospital. Protein Purification and Crystallization—Construction of the Mor expression plasmid, pIA69, has been described elsewhere (29Artsimovitch I. Activation of Middle Transcription of the Phage Mu. The University of Tennessee, Memphis1996Google Scholar). The plasmid contains the gene encoding an N-terminal histidine-tagged Mor protein (His-Mor; 17.1 kDa) under the control of a T7 promoter as well as a slightly modified Plac promoter we call PlacSYN (29Artsimovitch I. Activation of Middle Transcription of the Phage Mu. The University of Tennessee, Memphis1996Google Scholar, 30Zhao Z. Effects of Plys Promoter Sequence on Transcription Activation by the C Protein of Bacteriophage Mu. The University of Tennessee, Memphis1999Google Scholar). The His-Mor protein was overexpressed in E. coli strain JM109 DE3 (mcrA Δpro-lac thi gyrA96 endA1 hsdR17 relA1 supE44 recA λDE3/F′ lacIQ pro+; Promega Corp.). An overnight culture (200 ml) of a fresh transformant was transferred to 4 liters of LB medium containing 25 μg/ml chloramphenicol and grown at 37 °C until the A600 reached 0.5–0.6. The cells were induced with 1 mm IPTG for 3 h and harvested by centrifugation. Cell pellets were resuspended in 20 mm Tris-HCl, pH 7.9, 200 mm NaCl, 10% glycerol, 1 mm 2-mercaptoethanol and lysed by sonication. The His-Mor protein was purified by nickel-affinity chromatography (Qiagen). Buffer exchange into the storage buffer (20 mm Tris-HCl, pH 7.9, 50 mm NaCl, 10% glycerol, 1 mm EDTA, and 1 mm dithiothreitol) and further purification were achieved by gel filtration chromatography in a Superdex-75 column (Amersham Biosciences). For crystallization trials, the purified protein was concentrated to 50 mg/ml by using an Amicon YM30 (30-kDa cut-off) membrane filter, which retained the 34.2-kDa His-Mor dimer. Mass spectrometry and N-terminal sequencing confirmed that the purified protein was histidine-tagged Mor (data not shown). Crystallization was performed by the hanging drop diffusion method (31McPherson A. Preparation and Analysis of Protein Crystals. Krieger Publishing Co., Malabar, FL1989Google Scholar). Initial screening was carried out using Hampton Research screens and then refined. His-Mor crystals were obtained at either 4 or 18 °Cby the hanging drop diffusion method using 1.8–2.1 m NaCl as a precipitant in 0.1 m imidazole buffer (pH 7.0–7.2). Guanidine hydrochloride (125 mm) was used as an additive to improve the diffraction quality of the crystals. Because guanidine is a chaotrope, the secondary structure composition of the protein was analyzed by circular dichroism spectroscopy in the presence and absence of guanidine hydrochloride; no difference was observed between the two samples (data not shown). The crystals belong to space group P3221 with cell dimensions of a = 81.2 Å and c = 44.8 Å. There is one molecule in an asymmetric unit with a solvent content of 52%. Data Collection—Crystals were frozen in liquid nitrogen using a cryoprotectant solution made of the mother liquor supplemented with glycerol to a final concentration of 40%, and diffraction data were collected in a nitrogen gas stream (100 K). The mercury-derivative crystals were obtained by soaking in mother liquor with 10 mm thiomersal for 3 days at 4 °C. The two-wavelength data using mercury-derivative crystals and one-wavelength native data using a crystal grown in the absence of guanidine were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. The wavelengths for the data collection were determined from an x-ray fluorescence scan. We also prepared platinum derivatives by soaking the crystals with 10 mm K2PtCl4 for 3 days at 4 °C, and a single-wavelength data set was collected at the X-12C beamline at the National Synchrotron Light Source at Brookhaven National Laboratory. Mercury-derivative and guanidine-free crystals diffracted to respective resolutions of 2.6 and 2.5 Å, whereas platinum-derivative crystals diffracted to 2.0 Å resolution. Mercury-derivative data were processed and scaled using DENZO/SCALEPACK (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar), and the guanidine-free and the platinum-derivative data were processed with HKL2000 (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The data collection statistics are shown in Table I.Table IData collection and refinement statisticsMercury dataPlatinum dataPeakInflectionWavelength (Å)1.00511.00861.0721Resolution (Å)aLast shell value is in parentheses50—2.8 (2.85—2.8)50—2.8 (2.85—2.8)50—2.2 (2.24—2.2)Rmerge (%)aLast shell value is in parentheses,bRsym=Σhkl(Σi|Ihkl,i-〈Ihkl〉|)/Σhkl,i〈Ihkl〉, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l, and 〈Ihkl〉 is the mean intensity of that reflection7.5 (50.6)7.3 (48.1)5.5 (46.3)I/σaLast shell value is in parentheses22.1 (1.96)27.5 (2.37)41.8 (3.1)MAD phasing Resolution (Å)50—2.8 FOM0.52Anomalous scattering factors f′—18.518—13.983 f′9.6126.071Refinement Resolution (Å)aLast shell value is in parentheses30—2.2 (2.25—2.2) Protein atoms744 Nonprotein atoms25 Total reflections7746R-factor Working set/test setcRwork=Σ∥Fobs|-|Fcalc∥/Σ|Fobs|,where|Fobs|and|Fcalc| are observed and calculated structure factor amplitudes, respectively. Rfree is equivalent to Rwork except that 10% of the total reflections were set aside to test the progress of refinement0.252/0.268 (0.234/0.300) r.m.s.d. bonds (Å)dr.m.s.d. from ideal geometry0.010 r.m.s.d. angles (°)dr.m.s.d. from ideal geometry1.48Ramachandran plot (%) Most favored/allowed92.6/7.4a Last shell value is in parenthesesb Rsym=Σhkl(Σi|Ihkl,i-〈Ihkl〉|)/Σhkl,i〈Ihkl〉, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l, and 〈Ihkl〉 is the mean intensity of that reflectionc Rwork=Σ∥Fobs|-|Fcalc∥/Σ|Fobs|,where|Fobs|and|Fcalc| are observed and calculated structure factor amplitudes, respectively. Rfree is equivalent to Rwork except that 10% of the total reflections were set aside to test the progress of refinementd r.m.s.d. from ideal geometry Open table in a new tab Structure Determination—The structure was solved by the multi-wavelength anomalous dispersion method (33Hendrickson W.A. Ogata C.M. Methods Enzymol. 1997; 276: 494-523Crossref PubMed Scopus (326) Google Scholar) using two-wavelength data sets of mercury derivative; the peak wavelength was at 1.0051 Å, and the inflection wavelength was at 1.0086 Å. The single mercury site was located, and phase refinement was done by using the programs SOLVE and RESOLVE (34Terwilliger T. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 501-505Crossref PubMed Scopus (49) Google Scholar, 35Terwilliger T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1863-1871Crossref PubMed Scopus (199) Google Scholar). Initial phasing and model building was done with mercury-derivative data, and platinum-derivative data were used for subsequent refinement. Model building was done using the program O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) and refined with XPLOR (37Brunger A.T. X-PLOR: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar) and CNS (38Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Kunstleve R.W.G. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) to an Rfree of 0.268 and Rwork of 0.252 for all data in the resolution range of 20–2.2 Å. The mercury was bound to Cys61 of Mor, and platinum was bound to His63 and Met116 of Mor. The guanidine-free structure was refined to an Rfree of 0.311 and Rwork of 0.289 in the resolution range of 30–2.5 Å. Although these two heavy atoms were bound to different sites in the protein, conformational differences of the two derivative structures to the guanidine-free native structure were limited to the side chains of heavy atom-bound residues. This finding demonstrates that the two derivative structures basically are the same conformation as the guanidine-free native structure. The highest resolution platinum-derivative structure was used for structural interpretation. The refinement statistics of the platinum-derivative structure are summarized in Table I. Site-directed Mutagenesis of the Hydrophobic Core—The construction of the plasmids and bacterial strains used here were described elsewhere (25Artsimovitch I. Howe M.M. Nucleic Acids Res. 1996; 24: 450-457Crossref PubMed Google Scholar, 29Artsimovitch I. Activation of Middle Transcription of the Phage Mu. The University of Tennessee, Memphis1996Google Scholar). Plasmid pIA69, containing the mor gene with a histidine tag and silent restriction sites, was used as template for the PCR-based mutagenesis and cloning. Each mutagenic primer was designed to introduce multiple mutations at one position and synthesized using an equimolar mixture of desired nucleotides at the targeted position. The two strands were mutagenized in separate PCRs and then used as templates for overlapping PCR. The resulting mutagenized cassette was cloned into pIA69 between the PstI and HindIII sites. The ligation mixture was transformed into strain MH13435 (mcrA Δpro-lac thi gyrA96 endA1 hsdR17 relA1 supE44 recA/F′ lacIQI ΔlacZY pro+/pIA14), with a Pm-lacZ fusion reporter plasmid, pIA14 (25Artsimovitch I. Howe M.M. Nucleic Acids Res. 1996; 24: 450-457Crossref PubMed Google Scholar), and plated on LB plates with Ap and Cm. The resulting colonies were screened on MacConkey lactose agar indicator plates with Ap, Cm, and different concentrations of IPTG (0, 10, 50, 100, and 300 μm). Candidate mutant plasmids were chosen based on the plate phenotypes, and the mutations were identified by automated DNA sequencing. Mutant plasmids were transformed into MH13355 (mcrA Δpro-lac thi gyrA96 endA1 hsdR17 relA1 supE44 recA λDE3/F′ lacIQI ΔlacZY pro+); the λDE3 encodes T7 RNA polymerase for protein overproduction and purification (39Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). Crude Extract Preparation—Overnight cultures (2 ml) of MH13355 derivatives containing the mutant (and wild-type control) plasmids were transferred to 100 ml of LB containing Cm. The cells were grown at 37 °C until the A600 reached 0.4–0.6 and then induced with 1 mm IPTG for 60 min. After the cells were harvested by centrifugation, the cell pellets were resuspended in 3 ml of buffer M containing 20 mm Tris-HCl, pH 7.9, 200 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm 2-mercaptoethanol, and lysed by sonication. The sonicated preparation was subjected to centrifugation at 10,000 rpm for 15 min. The supernatant was removed, and the pellet was resuspended in 3 ml of buffer M. Samples of the supernatant (24 μl) and pellet (24 μl) were mixed with 6 μlof5× loading dye, boiled, and subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel (40Margolin W. Howe M.M. J. Bacteriol. 1990; 172: 1424-1429Crossref PubMed Google Scholar) stained with Coomassie Blue (41Wilson C.M. Methods Enzymol. 1983; 91: 236-247Crossref PubMed Scopus (141) Google Scholar), and the protein concentration was determined by a Bradford assay (42Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). In Vivo Transactivation Assay—Cells were grown overnight in 2 ml of M9CA medium containing Cm and Ap. A 50-μl sample of the overnight culture was inoculated into 10 ml of M9CA medium with the same antibiotics and grown at 37 °C until the A600 reached 0.4–0.6. A 2-ml sample was removed to serve as an uninduced control, and the remaining culture was induced with 2 mm IPTG for 60 min. Based on the plate phenotype of individual mutants, dilutions of the cells were made using M9CA medium, and the cells were permeabilized by mixing with 10 μl of chloroform and 18.5 μl of 0.1% SDS in a total volume of 100 μl. After incubation for 20 min on ice, 0.5 ml of o-nitrophenyl-β-galactopyranoside (0.833 mg/ml) in buffer Z (43Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-355Google Scholar) was added, and the mixture was incubated at 28 °C for 20 min. The reactions were stopped by adding 250 μl of 1 m Na2CO3, and spectrophotometer readings were taken at 420 nm for the reaction and 600 nm for the cell density. The β-galactosidase activities were calculated according to Miller's formula (43Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-355Google Scholar) and normalized relative to that of a wild-type culture assayed in parallel and set to 1000 Miller units. The crystal structure of His-Mor was solved by the multi-wavelength anomalous dispersion method using mercury-derivative crystals, and the refinement was done with the high resolution platinum-derivative data set. The final structure was refined to 2.2 Å resolution with an Rwork value of 26.4% and Rfree value of 24.8%. The asymmetric unit has one molecule. As expected, His-Mor forms a dimer, and the two subunits are related to each other by a crystallographic 2-fold symmetry axis. For the purpose of this discussion, the amino acid residues will be numbered according to their positions in the native protein. Out of the total 129 amino acids in native Mor, 26 residues
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