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• W2988893517 abstract "The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp–dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo l-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. In vitro TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp–dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif. The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp–dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo l-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. In vitro TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp–dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif. Translation termination in bacteria is initiated by a couple of protein paralogs named release factor 1 (RF1) 5The abbreviations used are: RFrelease factor1M-l-Trp1-methyl-l-TrpIPTGisopropyl β-d-1 thiogalactopyranosidel-PSA5′-O-(N-(l-prolyl)-sulfamoyl)-adenosinel-Trpl-tryptophanPTCpeptidyl transferase center. and 2 (RF2). Once bound to the ribosome, these RF proteins promote hydrolysis of the resident peptidyl-tRNA by aiding in the accommodation of a molecule of water at the ribosome active site, also known as the peptidyl transferase center (PTC) (1Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Crystal structure of a translation termination complex formed with release factor RF2.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (19064930): 19684-1968910.1073/pnas.0810953105Crossref PubMed Scopus (187) Google Scholar, 2Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Insights into translational termination from the structure of RF2 bound to the ribosome.Science. 2008; 322 (18988853): 953-95610.1126/science.1164840Crossref PubMed Scopus (232) Google Scholar). RF1 or RF2 initiates translation termination by recognizing distinct stop codons (UAA/UAG for RF1; UAA/UGA for RF2) located at the small subunit ribosomal A-site decoding center (3Scolnick E. Tompkins R. Caskey T. Nirenberg M. Release factors differing in specificity for terminator codons.Proc. Natl. Acad. Sci. U.S.A. 1968; 61 (4879404): 768-77410.1073/pnas.61.2.768Crossref PubMed Scopus (266) Google Scholar). Once these release factors recognize their corresponding stop codons, they change their free-state compact conformation (closed conformation) to an extended conformation (open conformation) where the tip of RF1/RF2 domain 3 reaches the PTC (4Trappl K. Joseph S. Ribosome induces a closed to open conformational change in release factor 1.J. Mol. Biol. 2016; 428 (26827724): 1333-134410.1016/j.jmb.2016.01.021Crossref PubMed Scopus (15) Google Scholar). The tip of the domain 3 of both proteins contains a conserved GGQ tripeptide motif whose methylated glutamine residue presumably aids in the positioning of a molecule of water (1Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Crystal structure of a translation termination complex formed with release factor RF2.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (19064930): 19684-1968910.1073/pnas.0810953105Crossref PubMed Scopus (187) Google Scholar, 5Shaw J.J. Green R. Two distinct components of release factor function uncovered by nucleophile partitioning analysis.Mol. Cell. 2007; 28 (17996709): 458-46710.1016/j.molcel.2007.09.007Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 6Mora L. Heurgue-Hamard V. de Zamaroczy M. Kervestin S. Buckingham R.H. Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.J. Biol. Chem. 2007; 282 (17932046): 35638-3564510.1074/jbc.M706076200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 7Pierson W.E. Hoffer E.D. Keedy H.E. Simms C.L. Dunham C.M. Zaher H.S. Uniformity of peptide release is maintained by methylation of release factors.Cell Rep. 2016; 17 (27681416): 11-1810.1016/j.celrep.2016.08.085Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Accommodation of the tip of domain 3, suggested to be the rate-limiting step during hydrolysis of the peptidyl-tRNA, requires several structural changes in the release factor protein as well as in the PTC (8Burakovsky D.E. Sergiev P.V. Steblyanko M.A. Kubarenko A.V. Konevega A.L. Bogdanov A.A. Rodnina M.V. Dontsova O.A. Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination.RNA. 2010; 16 (20668033): 1848-185310.1261/rna.2185710Crossref PubMed Scopus (20) Google Scholar, 9Svidritskiy E. Korostelev A.A. Conformational control of translation termination on the 70S ribosome.Structure. 2018; 26 (29731232): 821-828.e82310.1016/j.str.2018.04.001Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 10Korostelev A. Zhu J. Asahara H. Noller H.F. Recognition of the amber UAG stop codon by release factor RF1.EMBO J. 2010; 29 (20588254): 2577-258510.1038/emboj.2010.139Crossref PubMed Scopus (83) Google Scholar). RF2 promotes hydrolysis of peptidyl-tRNA three to six times more efficiently than RF1 (11Freistroffer D.V. Kwiatkowski M. Buckingham R.H. Ehrenberg M. The accuracy of codon recognition by polypeptide release factors.Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (10681447): 2046-205110.1073/pnas.030541097Crossref PubMed Scopus (130) Google Scholar). After hydrolysis of the peptidyl-tRNA, RF1 resides within the ribosomal complex until it dissociates from the ribosome with the help of release factor 3 (RF3), whereas RF2 can independently dissociate itself from the ribosomes (12Adio S. Sharma H. Senyushkina T. Karki P. Maracci C. Wohlgemuth I. Holtkamp W. Peske F. Rodnina M.V. Dynamics of ribosomes and release factors during translation termination in E. coli.Elife. 2018; 7 (29889659): e3425210.7554/eLife.34252Crossref PubMed Scopus (24) Google Scholar). RF1 protein has shown longer residence time within the ribosome than RF2, explaining the need for the release action of RF3 (12Adio S. Sharma H. Senyushkina T. Karki P. Maracci C. Wohlgemuth I. Holtkamp W. Peske F. Rodnina M.V. Dynamics of ribosomes and release factors during translation termination in E. coli.Elife. 2018; 7 (29889659): e3425210.7554/eLife.34252Crossref PubMed Scopus (24) Google Scholar), indicating that RF1 has stronger affinity for the ribosome than RF2 (13Zavialov A.V. Mora L. Buckingham R.H. Ehrenberg M. Release of peptide promoted by the GGQ motif of class 1 release factors regulates the GTPase activity of RF3.Mol. Cell. 2002; 10 (12419223): 789-79810.1016/S1097-2765(02)00691-3Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Translation termination is completed when the ribosome release factor interacts at the empty A-site, and along with elongation factor G disassembles the translating ribosome separating the two subunits from the translated mRNA and the last deacylated tRNA (14Pavlov M.Y. Freistroffer D.V. MacDougall J. Buckingham R.H. Ehrenberg M. Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3.EMBO J. 1997; 16 (9233822): 4134-414110.1093/emboj/16.13.4134Crossref PubMed Scopus (111) Google Scholar). release factor 1-methyl-l-Trp isopropyl β-d-1 thiogalactopyranoside 5′-O-(N-(l-prolyl)-sulfamoyl)-adenosine l-tryptophan peptidyl transferase center. The tnaCAB (tna) operon is a bacterial chromosomal unit whose function is related to the degradation of external l-tryptophan (l-Trp) and the formation of indole, a biofilm, and quorum-sensing regulatory metabolite (15Wood T.K. Hong S.H. Ma Q. Engineering biofilm formation and dispersal.Trends Biotechnol. 2011; 29 (21131080): 87-9410.1016/j.tibtech.2010.11.001Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The tna operon is constituted by a 5′-regulatory leader region and two structural genes tnaA and tnaB, which express the tryptophanase enzyme and an l-Trp–specific transporter, respectively. The 5′-regulatory leader region of the tna operon contains a small ORF, tnaC, and a Rho-dependent transcription termination sequence. Transcription of the tna operon is induced primarily by the absence of a rich carbon source (16Deeley M.C. Yanofsky C. Transcription initiation at the tryptophanase promoter of Escherichia coli K-12.J. Bacteriol. 1982; 151 (6284718): 942-951Crossref PubMed Google Scholar). Transcription coupled to translational expression of tnaC, which synthesizes the TnaC regulatory nascent peptide, allows RNA polymerase to continue synthesizing mRNA downstream of the tnaC gene. In Escherichia coli, under limiting concentrations of l-Trp, translation of the tnaC gene terminates at its UGA stop codon, releasing translating ribosomes from the nascent mRNA (17Gong F. Yanofsky C. Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.J. Biol. Chem. 2001; 276 (11050101): 1974-198310.1074/jbc.M008892200Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The release of the ribosomes exposes a rut site that overlaps the tnaC stop codon, allowing Rho to terminate transcription before reaching the first structural gene, tnaA. Under activating concentrations of l-Trp, ribosomes translating tnaC are arrested at its UGA stop codon, blocking the rut site and action of Rho and allowing transcription of the structural genes (18Stewart V. Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1986; 167 (3522554): 383-38610.1128/jb.167.1.383-386.1986Crossref PubMed Google Scholar, 19Gong F. Ito K. Nakamura Y. Yanofsky C. The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11470925): 8997-900110.1073/pnas.171299298Crossref PubMed Scopus (97) Google Scholar). Previous in vivo studies have shown that the expression of the tna structural genes is affected by the identity of the tnaC stop codon (20Konan K.V. Yanofsky C. Role of ribosome release in regulation of tna operon expression in Escherichia coli.J. Bacteriol. 1999; 181 (10049385): 1530-1536Crossref PubMed Google Scholar). Under the presence of activating concentrations of l-Trp, tnaC genes with either UAG or UAA codons induced less expression of tnaA’-’lacZ reporter genes than tnaC genes with UGA codons (20Konan K.V. Yanofsky C. Role of ribosome release in regulation of tna operon expression in Escherichia coli.J. Bacteriol. 1999; 181 (10049385): 1530-1536Crossref PubMed Google Scholar). Where 85 and 50% reporter gene expression reduction were observed after replacing the tnaC UGA stop codon with UAG and UAA stop codons, respectively (20Konan K.V. Yanofsky C. Role of ribosome release in regulation of tna operon expression in Escherichia coli.J. Bacteriol. 1999; 181 (10049385): 1530-1536Crossref PubMed Google Scholar). These observations suggest that the degree of inhibition of translation termination by l-Trp at the tnaC sequences may depend on the identity and kinetic characteristics of the release factor involved, with RF2, which recognizes the UGA stop codon, being more susceptible to l-Trp-induced inhibition of peptide release than RF1, which recognizes the UAG stop codon. Notably, these previous results were obtained in E. coli K12 strains that express a RF2 Thr-246 variant, a less active protein compared with the RF2 Ala-246 variant present in E. coli B strains (6Mora L. Heurgue-Hamard V. de Zamaroczy M. Kervestin S. Buckingham R.H. Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.J. Biol. Chem. 2007; 282 (17932046): 35638-3564510.1074/jbc.M706076200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Unfortunately, there are no studies of the expression of the tna operon under the action of the more active versions of RF2. Biochemical and structural analyses have shown that arrested ribosomes at the tnaC sequences contain TnaC-peptidyl-tRNAPro molecules in their P-sites (21Cruz-Vera L.R. Rajagopal S. Squires C. Yanofsky C. Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.Mol. Cell. 2005; 19 (16061180): 333-34310.1016/j.molcel.2005.06.013Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Bischoff L. Berninghausen O. Beckmann R. Molecular basis for the ribosome functioning as an L-tryptophan sensor.Cell Rep. 2014; 9 (25310980): 469-47510.1016/j.celrep.2014.09.011Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These studies also indicate that the TnaC nascent peptide acquires a specific conformation, induced by its interactions with the exit tunnel and free l-Trp molecules (22Bischoff L. Berninghausen O. Beckmann R. Molecular basis for the ribosome functioning as an L-tryptophan sensor.Cell Rep. 2014; 9 (25310980): 469-47510.1016/j.celrep.2014.09.011Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar23Martínez A.K. Gordon E. Sengupta A. Shirole N. Klepacki D. Martinez-Garriga B. Brown L.M. Benedik M.J. Yanofsky C. Mankin A.S. Vazquez-Laslop N. Sachs M.S. Cruz-Vera L.R. Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.Nucleic Acids Res. 2014; 42 (24137004): 1245-125610.1093/nar/gkt923Crossref PubMed Scopus (27) Google Scholar, 24Martínez A.K. Shirole N.H. Murakami S. Benedik M.J. Sachs M.S. Cruz-Vera L.R. Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.Nucleic Acids Res. 2012; 40 (22110039): 2247-225710.1093/nar/gkr1052Crossref PubMed Scopus (14) Google Scholar25Tian P. Steward A. Kudva R. Su T. Shilling P.J. Nickson A.A. Hollins J.J. Beckmann R. von Heijne G. Clarke J. Best R.B. Folding pathway of an Ig domain is conserved on and off the ribosome.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413621): E11284-E1129310.1073/pnas.1810523115Crossref PubMed Scopus (55) Google Scholar). Once l-Trp is removed, TnaC-tRNAPro molecules within the arrested ribosomes become sensitive to the hydrolysis action of RF2, indicating that l-Trp is required for the inhibition of the activity of RF2 at the PTC (26Cruz-Vera L.R. Gong M. Yanofsky C. Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide.Proc. Natl. Acad. Sci. U.S.A. 2006; 103 (16505360): 3598-360310.1073/pnas.0600082103Crossref PubMed Scopus (55) Google Scholar). Several studies indicate that conformational changes in the PTC induced through the TnaC nascent peptide and the exit tunnel components are important for inhibiting RF2 (22Bischoff L. Berninghausen O. Beckmann R. Molecular basis for the ribosome functioning as an L-tryptophan sensor.Cell Rep. 2014; 9 (25310980): 469-47510.1016/j.celrep.2014.09.011Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Cryo-EM structures of the ribosome in complex with the stalled TnaC nascent peptide show that the PTC nucleotide residues U2585 and A2602 acquire a conformational distribution that would clash with the amino acid residues at the tip of the domain 3 of RF2, likely hindering their proper positioning or accommodation within the ribosome (22Bischoff L. Berninghausen O. Beckmann R. Molecular basis for the ribosome functioning as an L-tryptophan sensor.Cell Rep. 2014; 9 (25310980): 469-47510.1016/j.celrep.2014.09.011Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 25Tian P. Steward A. Kudva R. Su T. Shilling P.J. Nickson A.A. Hollins J.J. Beckmann R. von Heijne G. Clarke J. Best R.B. Folding pathway of an Ig domain is conserved on and off the ribosome.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413621): E11284-E1129310.1073/pnas.1810523115Crossref PubMed Scopus (55) Google Scholar). Even though the structural studies revealed unfavorable conformational changes of PTC nucleotides, it remained unclear why the TnaC-mediated stalling could preferentially affect the function of RF2 over that of RF1, when their conserved GGQ motif acts in the same region of the ribosome during peptidyl-tRNA hydrolysis. In this work, we aimed to determine why the function of RF2 in translation termination is more susceptible to the inhibitory action of the TnaC nascent peptide and l-Trp in the ribosomal termination complex as compared with the function of RF1. We performed genetic and biochemical assays to study the effects of RF1 and RF2 protein variants on the expression of the tna operon, accumulation of arrested ribosomes, and cleavage of TnaC-tRNAPro2 molecules in the presence of l-Trp. Our results indicate that distinct RF2 residues located at the PTC during translation termination make this protein more susceptible than RF1 to translation termination inhibition by the TnaC nascent peptide and l-Trp. Previous experiments that compared the expression of tnaC tnaA’-’lacZ reporter genes containing variations in their tnaC stop codon (20Konan K.V. Yanofsky C. Role of ribosome release in regulation of tna operon expression in Escherichia coli.J. Bacteriol. 1999; 181 (10049385): 1530-1536Crossref PubMed Google Scholar) were performed under conditions where cellular l-Trp concentrations are affected by the expression of the tryptophanase enzyme (30Vazquez-Laslop N. Thum C. Mankin A.S. Molecular mechanism of drug-dependent ribosome stalling.Mol. Cell. 2008; 30 (18439898): 190-20210.1016/j.molcel.2008.02.026Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), making it difficult to compare the sensory capacity of these reporter genes for free l-Trp. We decided to analyze the in vivo expression of these tnaC tnaA’-’lacZ reporter gene variants using 1M-l-Trp as an inducer, because this analog of l-Trp can induce the expression of the tna operon without being metabolized by tryptophanase (27Yanofsky C. Horn V. Gollnick P. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.J. Bacteriol. 1991; 173 (1917834): 6009-601710.1128/jb.173.19.6009-6017.1991Crossref PubMed Scopus (0) Google Scholar). Bacterial cultures for each strain, SVS1144 (tnaC-UGA tnaA’-’lacZ) (28Stewart V. Yanofsky C. Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1985; 164 (3902796): 731-740Crossref PubMed Google Scholar), VK800 (tnaC-UAG tnaA’-’lacZ) (20Konan K.V. Yanofsky C. Role of ribosome release in regulation of tna operon expression in Escherichia coli.J. Bacteriol. 1999; 181 (10049385): 1530-1536Crossref PubMed Google Scholar), and PDG1144 (tnaC-UAA tnaA’-’lacZ) (29Gollnick P. Yanofsky C. tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.J. Bacteriol. 1990; 172 (2345136): 3100-310710.1128/jb.172.6.3100-3107.1990Crossref PubMed Google Scholar), were grown under several concentrations of 1M-l-Trp. It is important to note that these are K12-derived bacterial strains, and they therefore express the RF2 Thr-246 protein variant (6Mora L. Heurgue-Hamard V. de Zamaroczy M. Kervestin S. Buckingham R.H. Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.J. Biol. Chem. 2007; 282 (17932046): 35638-3564510.1074/jbc.M706076200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Bacterial pellets from the final cultures were used to determine β-gal activity (see “Experimental procedures”). Data are summarized in Fig. 1. We observed that the β-gal expression increased with increasing amounts of inducer in all three bacterial strains (Fig. 1A). At more than 40 μm 1M-l-Trp the SVS1144 (tnaC-UGA) strain generated the highest β-gal synthesis, two and five times more than the enzyme synthesis observed in the PDG1144 (tnaC-UAA) and VK800 (tnaC-UAG) strains, respectively. The graphs in Fig. 1A show a sharp increase in the enzyme expression between 0 and 5 μm 1M-l-Trp, which was previously observed with the SVS1144 strain (30Vazquez-Laslop N. Thum C. Mankin A.S. Molecular mechanism of drug-dependent ribosome stalling.Mol. Cell. 2008; 30 (18439898): 190-20210.1016/j.molcel.2008.02.026Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). After testing the range between 0 and 5 μm 1M-l-Trp, we observed a linear correlation between the expression of the enzyme and the concentration of inducer (Fig. 1B). We calculated that the reporter gene of the SVS1144 (tnaC-UGA) strain produced an expression efficiency of 230 Miller units of β-gal activity per 1 μm 1M-l-Trp, four times greater than the expression efficiency of the reporter gene of the PDG1144 (tnaC-UAA) strain and 20 times higher than the expression efficiency of the reporter gene of the VK800 (tnaC-UAG) strain. These results indicate that the reporter gene with a tnaC-UGA sequence is more sensitive to 1M-l-Trp than the constructs containing a tnaC sequence with either a UAA or a UAG codon. These data also suggest that 1M-l-Trp could be more efficient in blocking translation termination at the RF2-specific UGA codon than the RF1/2 UAA and RF1-specific UAG codons. The results shown in Fig. 1 suggest that l-Trp–induced ribosome arrest at the tnaC sequences could be more efficient if the tnaC stop codon is UGA rather than UAA or UAG. To determine whether such an argument could be true, we decided to analyze the formation in vitro of l-Trp–arrested ribosomes within the tnaC sequences containing variations at their stop codon positions. We used a reconstituted protein synthesis system obtained from E. coli K12 strains where T7 RNA polymerase transcribes supplied DNA fragments (PURExpress from New England Biolabs). The reconstituted protein synthesis system contains the RF2 Thr-246 protein variant. Reactions were performed using PCR fragments that contained the tnaC gene variants, where transcription is controlled by a T7 RNA polymerase promoter, and translation is enhanced by an omega sequence located upstream of the tnaC-Shine-Dalgarno ribosome-binding sequence (30Vazquez-Laslop N. Thum C. Mankin A.S. Molecular mechanism of drug-dependent ribosome stalling.Mol. Cell. 2008; 30 (18439898): 190-20210.1016/j.molcel.2008.02.026Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Northern blotting assays were performed to detect the accumulation of TnaC-peptidyl-tRNAPro2 (see “Experimental procedures”). Accumulation of TnaC-peptidyl-tRNAPro2 indicates that translation termination is inhibited during the expression of tnaC, generating arrested ribosomes at its stop codon. As seen in Fig. 2A, in the presence of 4 mm l-Trp, both 30 nm UGA and UAG tnaC-PCR fragments induced accumulation of TnaC-peptidyl-tRNAPro2 molecules (see lanes 1 and 3). To confirm that the detected signal is a product of the inhibitory action of l-Trp and the regulatory TnaC nascent peptide we used tnaC-PCR fragments containing a change at the tnaC Trp-12 codon (W12R). This amino acid substitution generates a nonfunctional TnaC nascent peptide that cannot produce ribosome arrest under activating concentrations of l-Trp (18Stewart V. Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1986; 167 (3522554): 383-38610.1128/jb.167.1.383-386.1986Crossref PubMed Google Scholar). We did not observe accumulation of TnaC-peptidyl-tRNAPro2 under the same conditions when the tnaC (W12R)-PCR fragments UGA and UAG were used for the reactions (compare lane 1 with 2 or lane 3 with 4), or when a translation inhibitor was added to the reactions (compare lanes 1 and 3 with lanes 5 and 7, respectively). These last sets of data suggest that such TnaC-peptidyl-tRNAPro2 accumulation depends on the amino acid composition of the TnaC nascent peptide and on the translation of both tnaC variants, UGA and UAG. We tested l-Trp–induced accumulation of TnaC-peptidyl-tRNAPro2 using several concentrations of tnaC-PCR fragments ranging from 0 to 60 nm to compare the capacity of both tnaC UGA and UAG variants for accumulating arrested ribosomes under high concentrations of l-Trp (4 mm). We observed that both tnaC variants generated maximum accumulation of TnaC-peptidyl-tRNAPro2 at DNA concentrations higher than 30 nm (Fig. 2B). We suspect that in the presence of 4 mm l-Trp, high concentrations of the tnaC PCR fragments could reduce the pool of tRNAPro2 limiting the production of stalled ribosomes, generating the plateau behavior observed in the curve obtained with the tnaC UGA PCR fragments (Fig. 2B, closed circles). Depletion of the cellular pool of tRNAPro2 has been seen in vivo during overexpression of the tnaC UGA gene (31Gong M. Gong F. Yanofsky C. Overexpression of tnaC of Escherichia coli inhibits growth by depleting tRNA2Pro availability.J. Bacteriol. 2006; 188 (16484200): 1892-189810.1128/JB.188.5.1892-1898.2006Crossref PubMed Scopus (23) Google Scholar). At concentrations lower than 30 nm, however, the tnaC-UAG DNA variant generated lesser amounts of TnaC-peptidyl-tRNAPro2 than the tnaC-UGA variant. The biggest differences between both variants were seen at concentrations lower than 7 nm, where the tnaC-UGA variant still reached maximum accumulation, whereas the tnaC-UAG variant produced roughly 40% of maximum accumulation. Therefore, we decided to do further in vitro tests using 7 nm for the two tnaC-PCR fragment variants. To determine the differences in l-Trp sensitivity of both constructs, we tested accumulation of TnaC-tRNAPro2 under two concentrations of l-Trp, 0.3 mm (low) and 4 mm (high), which have shown significant differences in in vitro accumulation of arrested ribosomes (27Yanofsky C. Horn V. Gollnick P. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.J. Bacteriol. 1991; 173 (1917834): 6009-601710.1128/jb.173.19.6009-6017.1991Crossref PubMed Scopus (0) Google Scholar). We observed that at 7 nm concentrations both the tnaC-UGA and tnaC–UAG DNA variants accumulated TnaC-peptidyl-tRNAPro2 under both high and low l-Trp concentrations (Fig. 2C). At high l-Trp concentrations the tnaC-UGA DNA generated twice the amount of TnaC-peptidyl-tRNAPro2 molecules than at low l-Trp concentrations. Similarly, at high l-Trp concentrations the tnaC-UAG variant generated four times more TnaC-peptidyl-tRNAPro2 molecules than at low l-Trp concentrations. Both results indicate that each of these tnaC variants are still sensitive to changes in l-Trp concentrations. However, the tnaC-UGA DNA variant accumulates more TnaC-peptidyl-tRNAPro2 than the tnaC-UAG DNA variant at either l-Trp concentrations (Fig. 2C, compare lanes 1 and 2 with lanes 5 and 6). At high l-Trp concentrations, the tnaC-UGA DNA variant accumulates four times more TnaC-peptidyl-tRNAPro2 molecules than the tnaC-UAG DNA variant (Fig. 2C, compare lane 2 with lane 6). Meanwhile, at low l-Trp concentrations, the tnaC-UGA DNA variant accumulates eight times more TnaC-peptidyl-tRNAPro2 molecules than the tnaC-UAG DNA variant (Fig. 2C, compare lane 1 with lane 5). These results suggest that the tnaC-UGA variant is more efficient at accumulating arrested ribosomes than the tnaC-UAG variant at the l-Trp concen" @default.
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• W2988893517 title "The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA" @default.
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