Role of Conserved Surface Amino Acids in Binding of SmpB Protein to SsrA RNA
2006; Elsevier BV; Volume: 281; Issue: 39 Linguagem: Inglês
10.1074/jbc.m605137200
ISSN1083-351X
AutoresDaniel P. Dulebohn, Hye Jin Cho, A. Wali Karzai,
Tópico(s)Bacteriophages and microbial interactions
ResumoBacteria possess a unique salvage mechanism for rescuing ribosomes stalled on aberrant mRNAs. A complex of SmpB protein and SsrA RNA orchestrates this salvage process. The specific and direct binding of SmpB facilitates recognition and delivery of SsrA RNA to stalled ribosomes. The SmpB protein is conserved throughout the bacterial kingdom and contains several conserved amino acid sequence motifs. We present evidence to demonstrate that amino acid residues Glu-31, Leu-91, and Lys-124, which are highly conserved and clustered along an exposed surface of the protein, play a crucial role in the SsrA-mediated peptide tagging process. Our analysis suggests that the peptide-tagging defect exhibited by these SmpB variants is due to their inability to facilitate the delivery of SsrA RNA to stalled ribosomes. Moreover, we present evidence to demonstrate that the ribosome association defect of these variants is due to their reduced SsrA binding affinity. Consistent with these findings, we present biochemical evidence to demonstrate that residues Glu-31, Leu-91, and Lys-124 are essential for the SsrA binding activity of SmpB protein. Furthermore, we have investigated the interactions of SmpB·SsrA orthologues from the thermophilic bacterium Thermoanaerobacter tengcongensis. Our investigations demonstrate an analogous role for the equivalent T. tengcongensis residues in SmpB·SsrA interactions, hence further validating our findings for the Escherichia coli SmpB·SsrA system. These results demonstrate the functional significance of this cluster of conserved residues in SmpB binding to SsrA RNA, suggesting they might represent a core contact surface for recognition of SsrA RNA. Bacteria possess a unique salvage mechanism for rescuing ribosomes stalled on aberrant mRNAs. A complex of SmpB protein and SsrA RNA orchestrates this salvage process. The specific and direct binding of SmpB facilitates recognition and delivery of SsrA RNA to stalled ribosomes. The SmpB protein is conserved throughout the bacterial kingdom and contains several conserved amino acid sequence motifs. We present evidence to demonstrate that amino acid residues Glu-31, Leu-91, and Lys-124, which are highly conserved and clustered along an exposed surface of the protein, play a crucial role in the SsrA-mediated peptide tagging process. Our analysis suggests that the peptide-tagging defect exhibited by these SmpB variants is due to their inability to facilitate the delivery of SsrA RNA to stalled ribosomes. Moreover, we present evidence to demonstrate that the ribosome association defect of these variants is due to their reduced SsrA binding affinity. Consistent with these findings, we present biochemical evidence to demonstrate that residues Glu-31, Leu-91, and Lys-124 are essential for the SsrA binding activity of SmpB protein. Furthermore, we have investigated the interactions of SmpB·SsrA orthologues from the thermophilic bacterium Thermoanaerobacter tengcongensis. Our investigations demonstrate an analogous role for the equivalent T. tengcongensis residues in SmpB·SsrA interactions, hence further validating our findings for the Escherichia coli SmpB·SsrA system. These results demonstrate the functional significance of this cluster of conserved residues in SmpB binding to SsrA RNA, suggesting they might represent a core contact surface for recognition of SsrA RNA. Specific complexes of RNA and protein perform many essential biological functions, including RNA processing, RNA turnover, RNA transport, and RNA folding as well as the translation of genetic information from mRNA into protein sequences. Principles that govern RNA-protein interactions are inadequately understood due in large part to a paucity of detailed structural and biochemical information on RNA-protein complexes. These principles are important for understanding RNA-protein machines, such as the ribosome and RNA-protein structure and function in general.SsrA (also known as transfer mRNA and 10Sa RNA) is a small, highly structured RNA that is found in all bacteria (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 2Keiler K.C. Shapiro L. Williams K.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7778-7783Crossref PubMed Scopus (149) Google Scholar, 3Withey J.H. Friedman D.I. Annu. Rev. Microbiol. 2003; 57: 101-123Crossref PubMed Scopus (126) Google Scholar). SsrA, through its unique sequence and structure, is endowed with both tRNA and mRNA-like functions (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 3Withey J.H. Friedman D.I. Annu. Rev. Microbiol. 2003; 57: 101-123Crossref PubMed Scopus (126) Google Scholar, 4Felden B. Hanawa K. Atkins J.F. Himeno H. EMBO J. 1998; 17: 3188-3196Crossref PubMed Scopus (110) Google Scholar, 5Zvereva M.E. Shpanchenko O.V. Dontsova O.A. Bogdanov A.A. Mol. Biol. 2000; 34: 1081-1089Crossref Scopus (2) Google Scholar, 6Zwieb C. Guven S.A. Wower I.K. Wower J. Biochemistry. 2001; 40: 9587-9595Crossref PubMed Scopus (18) Google Scholar, 7Gillet R. Felden B. Mol. Microbiol. 2001; 42: 879-885Crossref PubMed Scopus (81) Google Scholar, 8Grzymski E.C. Nat. Struct. Biol. 2003; 10: 321Crossref PubMed Scopus (3) Google Scholar, 9Keiler K.C. Waller P.R. Sauer R.T. Science. 1996; 271: 990-993Crossref PubMed Scopus (894) Google Scholar). The tmRNA model for SsrA function proposes that alaninecharged SsrA RNA recognizes stalled ribosomes, binds at the ribosomal A-site, and donates its alanine charge to the growing polypeptide (11Stepanov V.G. Nyborg J. Eur. J. Biochem. 2003; 270: 463-475Crossref PubMed Scopus (17) Google Scholar, 12Barends S. Karzai A. Sauer R. Wower J. Kraal B. J. Mol. Biol. 2001; 314: 9-21Crossref PubMed Scopus (92) Google Scholar, 13Rudinger-Thirion J. Giege R. Felden B. RNA. 1999; 5: 989-992Crossref PubMed Scopus (92) Google Scholar). The ribosomal reading frame then switches to the mRNA segment of SsrA, adding a degradation tag to the C terminus of targeted polypeptide. SsrA-tagged proteins are then recognized by cellular proteases and efficiently degraded (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 3Withey J.H. Friedman D.I. Annu. Rev. Microbiol. 2003; 57: 101-123Crossref PubMed Scopus (126) Google Scholar, 7Gillet R. Felden B. Mol. Microbiol. 2001; 42: 879-885Crossref PubMed Scopus (81) Google Scholar, 8Grzymski E.C. Nat. Struct. Biol. 2003; 10: 321Crossref PubMed Scopus (3) Google Scholar, 9Keiler K.C. Waller P.R. Sauer R.T. Science. 1996; 271: 990-993Crossref PubMed Scopus (894) Google Scholar). This unique bacterial process is also known as trans-translation. All known biological activities of SsrA RNA require small protein B (SmpB) (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 14Karzai A.W. Susskind M.M. Sauer R.T. EMBO J. 1999; 18: 3793-3799Crossref PubMed Scopus (265) Google Scholar). The known functions of SmpB are specific binding to SsrA RNA and promoting stable association and proper engagement of the SmpB·SsrA complex with stalled ribosomes (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 14Karzai A.W. Susskind M.M. Sauer R.T. EMBO J. 1999; 18: 3793-3799Crossref PubMed Scopus (265) Google Scholar, 15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar). In the absence of SmpB protein SsrA does not associate stably with 70 S ribosomes. SmpB binds specifically to the tRNA-like domain of SsrA and, although not required, enhances the efficiency of its aminoacylation by stabilizing SsrA tertiary structure (16Hanawa-Suetsugu K. Takagi M. Inokuchi H. Himeno H. Muto A. Nucleic Acids Res. 2002; 30: 1620-1629Crossref PubMed Scopus (76) Google Scholar). Therefore, formation of the SmpB·SsrA complex appears to be critical for recognition and rescue of stalled ribosomes.The SmpB protein is highly conserved in eubacteria. Sequence alignment analysis of SmpB orthologues from 115 bacterial species shows the presence of several conserved polar and hydrophobic residues. In studies described herein, we have sought to gain a better understanding of the biological role and energetic contributions of highly conserved surface amino acids of SmpB protein. We have specifically focused on a number of conserved residues that are closely clustered on one surface of the SmpB protein. We provide in vivo and in vitro evidence for the contribution of these amino acids in trans-translation. Additionally, we have investigated the SmpB·SsrA RNA complex of the thermophilic bacterium Thermoanaerobacter tengcongensis (Tten) 2The abbreviations used are: Tten, T. tengcongensis; N-Tricine, [2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type. 2The abbreviations used are: Tten, T. tengcongensis; N-Tricine, [2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type. and demonstrate that the equivalent residues make significant energetic contributions to the binding of Tten SmpB protein to Tten SsrA RNA. These results are consistent with the conclusion that this cluster of surface residues represents the core contact points of SmpB·SsrA interactions that is conserved across divergent bacterial species.MATERIALS AND METHODSSite-directed Mutagenesis—All single and double SmpB mutations were introduced by PCR mutagenesis using the Stratagene QuikChange kit. Plasmid pET28 BAHis6 (15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar) codes for SsrAHis6, SsrA RNA with a modified mRNA-like domain coding for a six-histidine tag. Escherichia coli strain W3110 ΔsmpB/(DE3) was transformed with individual pET28BAHis6 plasmid variants containing SmpBWT or one of the SmpB alanine variants, SmpBE31A, SmpBL91A, SmpBK124A, SmpBN93A, SmpBQ94A, SmpBE31A/L91A, SmpBE31A/K124A, SmpBL91A/K124A, SmpBN93A/Q94A, and SmpBE31A/L91A/K124A. Endogenous protein tagging assays were performed as previously described (15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar).Ribosome Association Assays—For 70 S ribosome preparations, 750-ml cultures of W3110 ΔsmpB/(DE3) containing plasmid pET28 BAWT or pET28BA with specified SmpB amino acid substitutions were grown in LB containing 3 μm isopropyl 1-thio-β-d-galactopyranoside to A600 of 0.8–1.0. Bacterial cells were harvested, washed in 50 mm Tris (pH 7.5), pelleted, and stored at –80 °C. Cell pellets were resuspended in buffer A (20 mm Tris (pH 7.5), 300 mm NH4Cl, 10 mm MgCl2, 0.5 mm EDTA, 6 mm β-mercaptoethanol, 10 units/ml SUPERase-In (Ambion)) and lysed by gentle sonication. Lysates were centrifuged at 33,000 × g for 30 min. Supernatants were transferred to new tubes and centrifuged again at 33,000 × g for 30 min. Typically, a 19-ml aliquot of the supernatant was layered onto a 32% sucrose cushion in buffer B (20 mm Tris (pH 7.5), 500 mm NH4Cl, 10 mm MgCl2, 0.5 mm EDTA, 6 mm β-mercaptoethanol, 10 units/ml SUPERase-In (Ambion)) and centrifuged at 85,000 × g for 22 h. The pellet containing tight-coupled ribosomes was washed twice with 5 ml of cold buffer B. Pellets were resuspended in 0.25 ml of buffer A, and equivalent numbers of ribosomes were loaded onto a 10–50% sucrose cushion in buffer A. The sucrose gradients were centrifuged at 42,000 × g for 17 h. Gradients were fractionated, and the fractions corresponding to purified 70 S ribosomes were pooled and used for Western and Northern blot analysis. RNA for Northern blot analysis was extracted with Tri-LS reagent (Molecular Research), and equal amounts of RNA were loaded onto 1% formaldehyde agarose gels, transferred to Hi-Blot nylon membrane (Amersham Biosciences), and probed with a psoralenbiotin (Ambion) labeled full-length SsrA probe. For Western blot analysis, an equal number of ribosomes were loaded per lane, and the associated proteins were resolved on 15% Tris-Tricine gels. Western blots were developed with antibodies raised against purified SmpB protein and a secondary IR 800-nm dye conjugated secondary antibody (Molecular Probes).Purification of SmpB Alanine-substituted Variants—Bacterial strain BL21 (DE3)/plysS (Stratagene) was transformed with plasmid pET28 BAHis6 harboring SmpBWT or one of the alanine variants: SmpBE31A, SmpBL91A, SmpBK124A, SmpBN93A, SmpBQ94A, SmpBE31A/L91A, SmpBE31A/K124A, SmpBL91A/K124A, SmpBN93A/Q94A, Tten-SmpBWT, Tten-SmpBE28A, Tten-SmpBL87A, Tten-SmpBH89A, Tten-SmpBR90A, or Tten-SmpBK120A. Typically, cells were grown in 3 liters of LB at 37 °C to an A600 of ∼0.5 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h. Cells were harvested and resuspended in lysis buffer (1 m NH4Cl, 150 mm KCl, 50 mm Tris (pH 8.0), 2 mm β-mercaptoethanol, 20 mm imidazole) and lysed by sonication (3 × 30-s pulses, with the addition of 0.1 ml of 0.1 m phenylmethylsulfonyl fluoride after each pulse). Cell lysates were centrifuged for 1 h at 15,000 rpm in an SS-34 rotor to remove cellular debris. Due to the greater thermal stability of the Tten SmpB protein, an additional heat treatment step was included for wild-type Tten SmpB protein and all of its alanine substitution variants. The S30 supernatants of Tten SmpB proteins were heated at 65 °C for 10 min. This treatment results in the denaturation and precipitation of greater than 80% of soluble E. coli proteins, whereas the Tten SmpB variants are entirely unaffected and remain soluble. The heat-treated protein samples were centrifuged for 1 h at 15,000 rpm to pellet the denatured E. coli proteins. The supernatant fractions were mixed with 2 ml of Ni2+-NTA resin (Qiagen) and pre-equilibrated in lysis buffer, and the binding reaction was permitted to proceed with gentle rocking for 1 h at4 °C. The nickel-nitrilotriacetic acid resin was applied to a chromatography column and washed 3× with 50 ml of lysis buffer. Proteins were eluted with 12 ml of elution buffer (150 mm KCl, 50 mm Tris (pH 8.0), 200 mm imidazole, 20 mm β-mercaptoethanol). The eluates were then diluted 3-fold in fast protein liquid chromatography buffer A (50 mm KCl, 50 mm Hepes (pH 7.5), 5 mm MgCl2, 2 mm β-mercaptoethanol) and applied onto a Mono S ion exchange column (Amersham Biosciences-GE Healthcare). A gradient of 50–850 mm KCl in buffer A was developed over 20 column volumes to isolate the SmpB protein. SmpB protein, with greater than 95% purity, elutes at ∼500 mm KCl under these conditions. Protein concentrations were determined by absorbance at 280 nm using extension coefficients of 29575 m–1 cm–1 for the E. coli SmpB variants and 10430 m–1 cm–1 for the Tten SmpB variants. Protein aliquots were stored at –80 °C until needed.Electrophoretic Mobility-shift Assays—SsrA variants were produced and labeled as described previously (15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar). The csrB gene was PCR-amplified from E. coli genomic DNA using a 5′-primer csrb2 (5′-CGAATTCTAATACGACTCACTATAGGTGTCTTCAGGACGAAGAAC-3′) and a 3′-primer csrb3(5′-AAAAGGGGTACTGTTTTACCAG-3′). The amplified product was purified by gel electrophoresis and re-amplified to incorporate a T7 promoter sequence at its 5′-end. CsrB RNA was transcribed using T7 RNA polymerase in accord with the manufacturer's recommendations (U. S. Biochemical Corp.). Template DNA was digested with DNase I, and the transcription products were phenol/chloroform-extracted and purified by electrophoresis on denaturing polyacrylamide gels. Electrophoretic mobility-shift assays were performed essentially as described (15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar), with minor modifications. Briefly, E. coli and T. tengcongensis SmpB protein variants were diluted to the desired protein concentrations in electrophoretic mobility-shift buffer (50 mm Tris (pH 7.5), 2 mm MgCl2, 300 mm KCl, 100 μg/ml bovine serum albumin, 2 mm β-mercaptoethanol 0.01% Nonidet P-40 (v/v), 10% glycerol (v/v)) in the presence of 100 nm CsrB RNA as a nonspecific competitor (in 100-fold excess over the specific SsrA RNA). Approximately 100 fmol of 3′-end-labeled SsrA113 RNA (∼1000 cpm/reaction) were added to each tube and incubated at room temperature for 30 min. Samples were loaded on a 12% non-denaturing gel and resolved by electrophoresis at 200 V in 0.5× Tris borate-EDTA to resolve the free RNA from RNA-protein complexes. Gels were run at 4 °C, dried, and exposed overnight to phosphorim-aging screens. All binding experiments for E. coli and T. tengcongensis SmpB variants were performed at least in triplicate, covering a protein concentration range of 0.1 nm to 1.5 μm. Data analysis was performed according to Berggrun and Sauer (17Berggrun A. Sauer R.T. J. Mol. Biol. 2000; 301: 959-973Crossref PubMed Scopus (7) Google Scholar). Briefly, the fraction of the primary bound species at each SmpB concentration was determined, and the apparent equilibrium dissociation constant was obtained by curve-fitting using the equation Θeq = C/(1 + Kd/[SmpB]i), where Θeq is the fraction of RNA bound at equilibrium, C is a constant representing the maximum fraction bound of the specific bound species, and [SmpB]i is the initial concentration of SmpB.RESULTSMutations in Conserved SmpB Amino Acids Decrease the Level of Tagged Proteins in Vivo—The SmpB protein is a requisite component of the trans-translation process. It performs several key functions, including specific binding to SsrA RNA, recognition of stalled ribosomes, and proper positioning of SsrA in the ribosomal A-site (1Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (340) Google Scholar, 3Withey J.H. Friedman D.I. Annu. Rev. Microbiol. 2003; 57: 101-123Crossref PubMed Scopus (126) Google Scholar, 14Karzai A.W. Susskind M.M. Sauer R.T. EMBO J. 1999; 18: 3793-3799Crossref PubMed Scopus (265) Google Scholar, 15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar). Sequence alignment analysis of SmpB protein revealed the identity of a number of invariant amino acid residues that are conserved across many diverse bacterial species (Fig. 1A). To gain insights into the functional significance of these highly conserved amino acids, we carried out a systematic alanine-scan mutagenesis of strategic residues of the E. coli SmpB protein. This analysis enabled us to evaluate the contribution of conserved residues to known SmpB functions in trans-translation. We previously reported on the functional significance of a number of highly conserved amino acid residues located in the C-terminal tail of the SmpB protein (15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar). In this study we focused specifically on several evolutionarily conserved amino acid residues (Glu-31, Leu-91, and Lys-124) and two marginally conserved residues (Asn-93, Q94), that are clustered along an exposed surface of the protein (Fig. 1C). We deemed alanine substitutions ideal for these studies, as alanine lacks a side chain beyond the β-carbon, does not impose steric restrictions, or alter the main-chain conformation. The mutants were generated by site directed mutagenesis using pET28BAHis6, a plasmid that harbors the smpB gene and a functional ssrA variant (ssrAHis6) that encodes ANDEHHHHHH in place of the normal ANDENYALAA degradation tag. The SsrAHis6 variant permits endogenously tagged proteins to be purified by nickel-nitrilotriacetic acid affinity chromatography and detected by Western blot analysis.To scrutinize the contributions of individual residues to known SmpB functions, we evaluated the affect of single alanine substitutions on the SmpB·SsrA mediated trans-translation process in vivo. To this end, we transformed an smpB deletion strain with the pET28BAHis6 plasmid, harboring a single alanine substitution mutant of SmpB. We purified endogenously His6 tagged products of the SmpB·SsrA system and resolved them by electrophoresis on SDS-polyacrylamide gels. The levels of endogenously tagged proteins were determined by Western blot analysis using anti-His6 antibodies. This analysis revealed that alanine substitutions of the two variably conserved SmpB residues (Asn-93 and Gln-94) did not have a substantial effect on the ability of SmpB to support tagging of endogenous substrates (data not shown). In contrast, alanine substitutions of SmpB residues Glu-31, Leu-91, and Lys-124 resulted in a modest and reproducible decrease in the level of endogenously tagged proteins. Compared with wild-type SmpB protein, these variants consistently displayed a 15–20% reduction in endogenous tagging activity, suggesting a role for these highly conserved residues in one of the known SmpB functions (data not shown).To gain further insight into the contributions of these residues, we generated a number of double alanine substitution variants of SmpB protein, including E31A/L91A (SmpBEL), E31A/K124A (SmpBEK), L91A/K124A (SmpBLK), and N93A/Q94A (SmpBNQ). Evaluation of the ability of the double alanine substitution variant SmpBNQ to support trans-translation in vivo revealed little or no loss of tagging activity. In contrast, double-alanine-substituted variants SmpBEL, SmpBLK, and SmpBEK showed a marked decrease in endogenous protein tagging activity (Fig. 2A, lanes 3–6). Compared with wild-type SmpB, the tagging propensity of these variants was reduced by 30, 50, and 65%, respectively (Fig. 2C). Maximum reduction in tagging activity was observed with a triple-alanine-substituted variant, E31A/L91A/K124A (SmpBELK), which showed a 70% loss of in vivo tagging activity (Fig. 2C). These findings demonstrate that several highly conserved residues, particularly residues Glu-31, Leu-91, and Lys-124, play an important role in trans-translation in vivo.FIGURE 2Analysis of the endogenous tagging activity of E. coli SmpB alanine substitution variants. A, a representative Western blot, developed with antibodies against the His6 epitope, displaying the endogenously tagged proteins. B, Coomassie-stained SDS-polyacrylamide gel showing that equal amounts of protein were loaded in each lane of panel A. C, bar graphs, representing the mean and S.D. of five independent tagging assays, display the level of tagging activity by the indicated SmpB variants, as compared with wild-type SmpB protein. Wild-type SmpB was used as a positive control, and SmpB59, a nonfunctional truncated SmpB variant, was used as a negative control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This Cluster of Conserved Residues Is Important for the in Vivo Association of the SmpB·SsrA Complex with 70 S Ribosomes—Having established that the highly conserved residues Glu-31, Leu-91, and Lys-124 play a key role in trans-translation, we wished to ascertain whether the decrease in endogenous tagging activity was due to a compromised ability of the SmpB variants to facilitate association of the SmpB·SsrA complex with stalled ribosomes. We have previously shown that the SmpB protein is required for stable association of SsrA RNA with 70 S ribosomes (14Karzai A.W. Susskind M.M. Sauer R.T. EMBO J. 1999; 18: 3793-3799Crossref PubMed Scopus (265) Google Scholar, 15Sundermeier T.R. Dulebohn D.P. Cho H.J. Karzai A.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2316-2321Crossref PubMed Scopus (69) Google Scholar). To assess the ribosome association propensities of the variants, we purified 70 S ribosomes from ΔsmpB cells harboring wild-type SmpB or one of its variants (SmpBEL, SmpBEK, SmpBLK, and SmpBELK) and measured the levels of associated SmpB protein by Western blot analysis. This assessment revealed that all of the double- and triple-alanine-substituted SmpB variants had substantially decreased levels of SmpB protein associated with 70 S ribosomes (Fig. 3A, lanes 3–6). The most notable decrease was observed with double alanine variant SmpBEK and the triple alanine variant SmpBELK (Fig. 3C).FIGURE 3Ribosome association assays. A, Western blot analysis with anti-SmpB antibodies displaying the amount of wild-type SmpB and select SmpB alanine substitution variants associated with 70 S ribosomes in vivo. The SmpB variant expressed in cells from which the ribosomes were purified is indicated on top. B, Coomassie stained SDS-polyacrylamide of samples in panel A, demonstrating that equal amounts of total protein were loaded in each lane. C, bar graphs, representing the mean and S.D. of three independent experiments, display the level of each SmpB variant associated with purified 70 S ribosomes, as compared with wild-type SmpB protein. Wild-type SmpB was used as a positive control, and SmpB59, a nonfunctional truncated SmpB variant, was used as a negative control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)One possible explanation for these findings could be that the substituted proteins are less stable and/or aggregate in the cell. This would lead to lower amounts of SmpB protein available to interact with SsrA RNA and mediate its association with 70 S ribosomes. To examine this possibility, we analyzed the levels of each protein in total cellular extracts and the S30 soluble fractions. We found that, like wild-type SmpB, the alanine-substituted proteins did not aggregate and were not present in the pellet fractions (data not shown). We found the SmpBE31A, SmpBL91A, SmpBK124A, SmpBEL, SmpBEK, and SmpBLK variants to be soluble, as wild-type levels of these substituted proteins were present in S30 extracts (Fig. 4, lanes 3–6, and data not shown). These results demonstrate that the ribosome association defects attributed to these variants were not due to a decrease in the total amount of SmpB protein present in the cells. These findings do suggest, however, that alanine substitutions of these highly conserved residues deter stable association of the SmpB protein with 70 S ribosomes.FIGURE 4Analysis of the expression level of SmpB alanine substitution variants. A, Western blot analysis with anti-SmpB antibodies showing the amount of each SmpB variant present in the soluble S30 extract. B, Coomassie-stained SDS-PAGE of the 30S extract of the samples, demonstrating that equal amounts of protein were loaded per lane in panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because SmpB function is required for stable association of SsrA RNA with stalled ribosomes, we probed for the amount of SsrA RNA in the same 70 S ribosome preparations. Consistent with our findings for SmpB protein, the level of 70 S ribosome-associated SsrA RNA was diminished in all SmpB variants tested (Fig. 5). The greatest decrease in the level of ribosome associated SsrA RNA was observed with SmpBELK followed by SmpBEK, SmpBLK, and SmpBEL (Fig. 5, A and C). This order corresponds precisely to the observed endogenous tagging phenotypes of these variants. Taken together, these data suggest that the ribosome association defects of the SmpB variants and the dramatic decrease in the levels of ribosome-associated SsrA RNA are most likely responsible for the deficiencies observed in the endogenous tagging assays (Fig. 2).FIGURE 5Ribosome association assays. A, Northern blot analysis with an SsrA RNA-specific probe to detect 70 S ribosome associated SsrA RNA. B, ethidium bromide staining of the same gel as in A, shown to demonstrate that similar amounts of ribosomal RNA, were loaded in each lane. The SmpB variant expressed in cells from which the ribosomes were purified is indicated on top. C, bar graphs, representing the mean and S.D. of three independent experiments, display the level of SsrA RNA associated with 70 S ribosomes purified from cells expressing the indicated SmpB variants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Alanine Substitution Variants of E. coli SmpB Protein Exhibit Defects in Binding SsrA RNA in Vitro—A critical first step in trans-translation is the binding of SmpB protein to SsrA RNA. Formation of a stable SmpB·SsrA complex is critical for the facilitated delivery of SsrA RNA to stalled ribosomes. Thus, the ability of SmpB to deliver SsrA to stalled ribosomes is directly related to the binding affinity and specificity of SmpB protein for SsrA RNA. To characterize this step in trans-translation, we generated expression constructs of SmpB protein carrying single, double, and triple alanine substitutions of key amino acid residues. Expression and purification of these substituted proteins followed the same basic procedure as for wild-type SmpB protein (see "Materials and Methods"). In all cases the substituted proteins were purified to greater than 95% homogeneity as detected by Coomassie-stained SDS-polyacrylamide gels. The apparent equilibrium dissociation constants (Kd) o
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