Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp–directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues. Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp–directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues. Ribosome arresting peptides (RAPs) are nascent polypeptides that act in cis on the translating ribosome to control the expression of genes by inducing ribosome arrest during translation elongation or termination. RAPs commonly sense external forces or low molecular weight compounds in the environment that spatially and temporally contribute to the expression of genes. RAPs such as SecM (1Nakatogawa H. Ito K. Secretion monitor, SecM, undergoes self-translation arrest in the cytosol.Mol. Cell. 2001; 7: 185-192Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) that sense external forces on the ribosome are typically large, because these nascent peptides have a domain that functions outside of the ribosome. In contrast, those that sense small molecules inside of the ribosome, such as TnaC are smaller (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar, 3Su T. Kudva R. Becker T. Buschauer R. Komar T. Berninghausen O. et al.Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.Nucleic Acids Res. 2021; 49: 9539-9547Crossref PubMed Scopus (8) Google Scholar). Typically, larger RAPs interact with cellular factors that can control their capacity for arresting ribosomes (4Butkus M.E. Prundeanu L.B. Oliver D.B. Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation.J. Bacteriol. 2003; 185: 6719-6722Crossref PubMed Scopus (84) Google Scholar, 5Fujiwara K. Ito K. Chiba S. MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome.Sci. Rep. 2018; 8: 10311-10322Crossref PubMed Scopus (9) Google Scholar, 6Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (47) Google Scholar). Because of their size and proximity to ribosomal components, large RAPs clearly show two structural domains, a sensor domain and an arresting domain. At the moment of the arrest for the large RAPs, the sensor domain is located outside the ribosome exit tunnel (4Butkus M.E. Prundeanu L.B. Oliver D.B. Translocon "pulling" of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation.J. Bacteriol. 2003; 185: 6719-6722Crossref PubMed Scopus (84) Google Scholar, 5Fujiwara K. Ito K. Chiba S. MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome.Sci. Rep. 2018; 8: 10311-10322Crossref PubMed Scopus (9) Google Scholar, 6Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (47) Google Scholar), whereas the arresting domain remains inside the tunnel (5Fujiwara K. Ito K. Chiba S. MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome.Sci. Rep. 2018; 8: 10311-10322Crossref PubMed Scopus (9) Google Scholar, 6Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (47) Google Scholar, 7Nakatogawa H. Ito K. The ribosomal exit tunnel functions as a discriminating gate.Cell. 2002; 108: 629-636Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). The short RAPs currently characterized interact with the compounds that they sense by using the ribosome exit tunnel as a binding surface (8Dever T.E. Ivanov I.P. Sachs M.S. Conserved upstream open reading frame nascent peptides that control translation.Annu. Rev. Genet. 2020; 54: 237-264Crossref PubMed Scopus (49) Google Scholar). For these short RAPs, it has been determined that conserved amino acid residues are necessary to induce arrest (9Ito K. Chiba S. Arrest peptides: cis-acting modulators of translation.Annu. Rev. Biochem. 2013; 82: 171-202Crossref PubMed Scopus (192) Google Scholar) by either directly binding the effector molecule or by acting at the peptidyl-transferase center (PTC) during ribosome arrest (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar, 3Su T. Kudva R. Becker T. Buschauer R. Komar T. Berninghausen O. et al.Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.Nucleic Acids Res. 2021; 49: 9539-9547Crossref PubMed Scopus (8) Google Scholar, 10Herrero Del Valle A. Seip B. Cervera-Marzal I. Sacheau G. Seefeldt A.C. Innis C.A. Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.Nat. Microbiol. 2020; 5: 554-561Crossref PubMed Scopus (32) Google Scholar, 11Arenz S. Ramu H. Gupta P. Berninghausen O. Beckmann R. Vazquez-Laslop N. et al.Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide.Nat. Commun. 2014; 5: 3501-3508Crossref PubMed Scopus (103) Google Scholar). However, because the size of short RAPs ranges from only a few to a couple of dozen amino acids, as in the case of TnaC, it has remained unclear whether short RAPs are constituted by the two independent sensor and stalling domains, as it has been observed with larger RAPs. In Escherichia coli, extracellular free L-tryptophan (L-Trp) is transported (12Yanofsky C. Horn V. Gollnick P. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.J. Bacteriol. 1991; 173: 6009-6017Crossref PubMed Scopus (0) Google Scholar) and catabolized into indole, pyruvate, and ammonia (13Newton W.A. Snell E.E. Catalytic Properties of tryptophanase, a multifunctional pyridoxal phosphate enzyme.Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 382-389Crossref PubMed Scopus (126) Google Scholar) by proteins encoded by the tnaCAB operon (tna) (14Deeley M.C. Yanofsky C. Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.J. Bacteriol. 1981; 147: 787-796Crossref PubMed Google Scholar). The E. coli tna operon's transcript is organized into two functional segments: the first segment is comprised of a 5′-regulatory leader region containing the tnaC RAP gene and a rut site that serves as a binding site for the Rho transcription termination factor. The second segment contains the tna structural genes, tnaA and tnaB, which encode the L-Trp catabolic enzyme tryptophanase and an L-Trp transporter, respectively (15Gong F. Yanofsky C. Instruction of translating ribosome by nascent peptide.Science. 2002; 297: 1864-1867Crossref PubMed Scopus (205) Google Scholar, 16Stewart V. Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1986; 167: 383-386Crossref PubMed Google Scholar). Transcription initiation is controlled by catabolite repression (17Botsford J.L. DeMoss R.D. Catabolite repression of tryptophanase in Escherichia coli.J. Bacteriol. 1971; 105: 303-312Crossref PubMed Google Scholar), while the continuation of transcription into the structural genes is regulated by nascent TnaC peptides through an L-Trp–dependent antitermination mechanism (18Gong 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: 8997-9001Crossref PubMed Scopus (104) Google Scholar, 19Stewart V. Yanofsky C. Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1985; 164: 731-740Crossref PubMed Google Scholar). Under glucose-limiting conditions, transcription initiates at the tna promoter and continues until reaching intrinsic pause sites found within the untranslated spacer region (20Gong F. Yanofsky C. A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region.J. Bacteriol. 2003; 185: 6472-6476Crossref PubMed Scopus (28) Google Scholar). The paused polymerase permits translation of tnaC to begin and serves to synchronize the transcription of the structural genes with the translation of the leader region (20Gong F. Yanofsky C. A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region.J. Bacteriol. 2003; 185: 6472-6476Crossref PubMed Scopus (28) Google Scholar). Transcriptional termination at the 5′-regulatory leader region of the tna operon depends on changes in L-Trp concentrations that are sensed by the TnaC RAP within the ribosome. During translation, TnaC monitors for these fluctuations, by folding into two α-helical domains. Formation of the helical domains allows TnaC to compact and form an L-Trp–specific binding site (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar, 3Su T. Kudva R. Becker T. Buschauer R. Komar T. Berninghausen O. et al.Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.Nucleic Acids Res. 2021; 49: 9539-9547Crossref PubMed Scopus (8) Google Scholar). In conditions of low L-Trp, the tnaC UGA stop codon enters the translating ribosome A-site where release factor 2 (RF2) induces hydrolysis of the resident TnaC-peptidyl-tRNAPro, resulting in translation termination (18Gong 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: 8997-9001Crossref PubMed Scopus (104) Google Scholar). The separation of the two ribosomal subunits then exposes the rut site that is positioned in the untranslated spacer immediately adjacent the tnaC stop codon (21Gong 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: 1974-1983Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and Rho-dependent transcription termination (attenuation) ensues before transcribing the downstream catabolic genes (21Gong 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: 1974-1983Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Alternatively, when L-Trp is in abundance, the compacted TnaC nascent chain will bind L-Trp and block RF2-mediated hydrolysis of the resident TnaC-peptidyl-tRNAPro, leaving the arrested ribosome tethered to the mRNA (18Gong 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: 8997-9001Crossref PubMed Scopus (104) Google Scholar, 22Cruz-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: 333-343Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The arrested ribosome blocks access of Rho to the rut site and transcription continues downstream expressing the structural genes (21Gong 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: 1974-1983Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). TnaC contains four highly conserved amino acid residues that are critical for the induction of ribosome arrest by free L-Trp, namely W12, D16, I19, and P24, and mutations at these residues abolish L-Trp–dependent induction (16Stewart V. Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.J. Bacteriol. 1986; 167: 383-386Crossref PubMed Google Scholar, 23Cruz-Vera L.R. Yanofsky C. Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of tna operon expression.J. Bacteriol. 2008; 190: 4791-4797Crossref PubMed Scopus (43) Google Scholar, 24Martinez A.K. Gordon E. Sengupta A. Shirole N. Klepacki D. Martinez-Garriga B. et al.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: 1245-1256Crossref PubMed Scopus (29) Google Scholar). A 2.6 Å cryo-EM structure of the TnaC-tRNAPro-ribosome arrested complex identified W12 and D16 residues near the narrow region of the ribosome exit tunnel (known as constriction region) to be involved in binding a single free L-Trp molecule in conjunction with 23S rRNA nucleotides and the K90 residue of the ribosomal uL22 protein (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar, 3Su T. Kudva R. Becker T. Buschauer R. Komar T. Berninghausen O. et al.Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.Nucleic Acids Res. 2021; 49: 9539-9547Crossref PubMed Scopus (8) Google Scholar). The uL22 K90 residue was previously identified by mutational changes to be important for L-Trp–induced expression of the tna operon (22Cruz-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: 333-343Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 25Martinez 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: 2247-2257Crossref PubMed Scopus (15) Google Scholar). This structure shows that TnaC folds into two distinct short α-helices connected by a hinge-like configuration with the W12 residue encompassed by the first α-helical structure (α1) at the constriction region. The second α-helix (α2) is close to the PTC and holds the I19 and P24 residues. The hinge is centered on the side chain of the I19 residue and positions the D16 residue at the top of the binding site. This unique structural distribution has not been observed in currently known structures of other large or short RAPs (Fig. 1) and suggests that TnaC might be structured with two independent functional domains. In this report, we compared the ability of several TnaC mutant peptides to induce ribosome arrest in response to L-Trp. Using reporter genes, we tested in vivo the suppressor capacity of two changes at nonconserved residues located in the α1 and α2 TnaC helixes, S10P and R23H, respectively. We tested the effects of these mutations to suppress loss-of-function (LOF) mutations at conserved residues W12, D16, and P24. Furthermore, we evaluated the L-Trp sensitivity in vitro of TnaC peptides containing either S10P or R23H. We show in this work that the nature of nonconserved residues in TnaC affects its capacity to sense L-Trp and arrest ribosomes during translation through two independent functional domains. We have shown previously that mutating the nonconserved TnaC R23 residue partially restores the capacity for inducing ribosome arrest by the LOF TnaC(D16E) mutant peptide in the presence of L-Trp (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). Changes at this residue (R23H and R23F) located nearby the PTC reduce the kinetics for accommodating puromycin and RF2 at the PTC, increasing the opportunity of L-Trp to bind and induce ribosome arrest (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). We tested whether changes at other nonconserved positions of TnaC would produce suppressor effects similar to those induced by R23 changes. We decided to continue isolating suppressors for the LOF TnaC(D16E) mutant peptide through the strategy used previously (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). We identify a new suppressor mutation at the TnaC S10 residue, S10P. The TnaC S10 residue is positioned at the exit tunnel near the L-Trp binding site, far from the PTC (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). Our results showed that the S10P change partially suppressed the D16E noninducible phenotype. As seen in Table 1, the tnaA'-'lacZ reporter construct controlled by the tnaC(D16E/S10P) mutant in cells grown in the presence of L-Trp showed ∼10-fold higher β-galactosidase (β-gal) levels than those levels observed with the reporter gene controlled by the tnaC(D16E) mutant (compare row 5 with row 3, third column). In the absence of added L-Trp, the tnaC(D16E/S10P) reporter expressed ∼4-fold higher β-gal levels than the tnaC(D16E) reporter (Table 1 compare row 5 with row 3, second column). The tnaC(S10P) mutant alone did not affect the expression of the reporter gene in the presence of L-Trp; that is, we did not observed differences in the expression between the reporter constructs with tnaC(WT) and tnaC(S10P) (Table 1, compare row 4 with row 2, third column). However, the tnaC(S10P) mutant had a profound effect on the reporter gene expression when cells were cultured in the absence of L-Trp. The reporter gene with the tnaC(S10P) variant showed in absence of L-Trp ∼20-fold higher β-gal levels than its counterpart with the WT tnaC variant (Table 1, compare row 4 with row 2, second column). These data show that the S10P change by itself substantially increased expression of the tnaA'-'lacZ reporter gene in the absence of exogenously added L-Trp.Table 1S10P and R23H are cis-acting mutations that suppress the LOF D16E mutationStrainβ-gal activity (MU)aCultures of Escherichia coli bacterial strains AW153 (WT), AW821 (D16E), AW888 (S10P), AW909 (D16E/S10P), AW643 (WT(ΔAUG)), AW940 (S10P (ΔAUG)), AW946 (D16E (ΔAUG)), and AW949 (S10P/D16E (ΔAUG)) were grown in M9-MM plus 0.2% glycerol, 0.05% ACH, 0.01% vitamin B1, with (+Trp) or without (-Trp) 100 μg/ml L-Trp. β-gal assays were performed in three independent experiments.Induction ratio (+Trp/-Trp)bInduction ratio of values for cultures grown with L-Trp (+Trp) and those cultured without L-Trp (-Trp).-Trp+TrpWT57 ± 22175 ± 27438.2D16E38 ± 1168 ± 251.8S10P1312 ± 2902506 ± 2441.9S10P/D16E138 ± 14568 ± 1374.1WT (ΔAUG)7 ± 17 ± 01.0D16E (ΔAUG)7 ± 17 ± 01.0S10P (ΔAUG)8 ± 08 ± 21.0S10P/D16E (ΔAUG)10 ± 09 ± 10.9Abbreviations: ACH, casein acid hydrolyzed; β-gal, β-galactosidase; LOF, loss-of-function; L-Trp, L-tryptophan; M9-MM, M9 minimal medium; MU, Miller units.a Cultures of Escherichia coli bacterial strains AW153 (WT), AW821 (D16E), AW888 (S10P), AW909 (D16E/S10P), AW643 (WT(ΔAUG)), AW940 (S10P (ΔAUG)), AW946 (D16E (ΔAUG)), and AW949 (S10P/D16E (ΔAUG)) were grown in M9-MM plus 0.2% glycerol, 0.05% ACH, 0.01% vitamin B1, with (+Trp) or without (-Trp) 100 μg/ml L-Trp. β-gal assays were performed in three independent experiments.b Induction ratio of values for cultures grown with L-Trp (+Trp) and those cultured without L-Trp (-Trp). Open table in a new tab Abbreviations: ACH, casein acid hydrolyzed; β-gal, β-galactosidase; LOF, loss-of-function; L-Trp, L-tryptophan; M9-MM, M9 minimal medium; MU, Miller units. At least two different mechanisms might explain the high basal level expression of lacZ observed with the S10P mutant in absence of L-Trp. One possibility is that changes in the S10 codon affect tnaC mRNA secondary structure leading to inefficient transcription termination of the tnaC regulatory region. A second possibility is that the TnaC(S10P) mutant RAP alone induces ribosome arrest at the low L-Trp levels found in vivo in the absence of exogenously added L-Trp. To discern between these possible scenarios, we tested in vivo the expression of our reporter gene with WT and mutant tnaC variants with or without their tnaC-AUG start codon. If the mutant RAP causes the high basal levels of the reporter gene, then in the absence of its translation (absence of the start codon), premature transcription termination will occur in the reporter gene leading to a drastic decrease in -gal expression in either the presence or absence of L-Trp. As expected, the absence of translation of WT tnaC dramatically decreased the expression of lacZ both in the presence or absence of additional L-Trp (Table 1, compare row 6 with row 2). Likewise, the expression of the reporter gene was also dependent on the translation of the TnaC(S10P) (Table 1, compare row 8 with row 4) and TnaC(D16E/S10P) (Table 1, compare row 9 with row 5) mutant variants. The data obtained with tnaC(S10P) mutant variants is similar to those observed with tnaC(R23H) mutants (Table S3) (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). These results indicate that the effects of the S10P change are dependent on translation of the mutant TnaC peptide and do not reflect effects exerted by structural changes in the RNA sequence affecting transcription termination. Taken together, these data indicate that the S10P change could promote similar effects at the PTC of the TnaC–ribosome complex as observed with the R23 variants (2van der Stel A.X. Gordon E.R. Sengupta A. Martinez A.K. Klepacki D. Perry T.N. et al.Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling.Nat. Commun. 2021; 12: 5340Crossref PubMed Scopus (17) Google Scholar). Alternatively, the S10P mutation could alter the sensitivity of TnaC for L-Trp without affecting the PTC. To confirm and extend the findings obtained using lacZ reporter assays, we generated a plasmid construct to analyze cell survival based on TnaC expression. A tnaC tnaA'-'camR reporter construct was produced by replacing the transcriptional regulatory region of the chloramphenicol resistance camR gene encoding chloramphenicol acetyl-transferase (CAT) in pACYC184 with the 5'-regulatory region of the tna operon (Experimental procedures). Thus, with this reporter, CAT protein expression is under the control of TnaC and free L-Trp. Initially, we monitored camR expression of our constructs using an enzymatic assay that measures the concentration of free sulfhydryls that result from CAT activity (Experimental procedures). Cell lysate from cultures grown in the presence of L-Trp produced CAT rate of reaction of 0.037 U, while extracts from cells grown without L-Trp produced a rate of reaction = 0.01 U. These results indicate a ≈3.7-fold increase in CAT activity of cells grown with L-Trp compared to those cells that were not cultured with exogenous L-Trp (Fig. 2A, compare closed diamonds with open diamonds, respectively). We concluded that WT TnaC peptides induce camR expression in the presence of L-Trp. Levels of CAT expression in cells exposed to L-Trp was similar to that in cells containing the natural camR promoter (pACYC plasmid) (Fig. 2A, compare closed circles with closed diamonds), suggesting that both regulatory regions tna and camR have similar transcriptional and translational efficiencies. In parallel to the CAT enzymatic assays, we also performed, cell growth challenges with chloramphenicol (Cm) to calculate the inhibitory concentration for reducing 50% (IC50) and 90% (IC90) of growth in cultures of E. coli harboring the tnaC tnaA'-'camR reporter (Experimental procedures). Corresponding to what was observed with the CAT enzymatic assays, we observed that cell cultures containing WT tnaC reporter plasmids were more resistant to Cm in the presence of L-Trp than in its absence (Fig. 2B and Table 2, row 2). These data show that the tnaC tnaA'-'camR reporter gene construct can be used to analyze and quantify the L-Trp sensing capacity of the 5'-tna regulatory region by determining Cm-IC values of cell growth and CAT activity levels.Table 2IC50 and IC90 values of cell growth of bacteria with tnaC tnaA'-'camR reporter gene fusion variantstnaC gene-Trp+TrpaCultures were grown in M9-MM with (+Trp) or without (-Trp) 20 μM 1MTrp as indicated in Experimental procedures.IC50bIC values in μg/ml were estimated using the plots shown at Figure 1 and supporting information (Figs. 1, S3 and S5).IC90IC50IC90wt<120170<170<IC<220270<S10P170<IC<220320<170<IC<220320<R23H170<IC<220320<170<IC<220320<D16 changes D16E<120<120<120<120S10P/D16E120<IC<170220<120<IC<170220< D16E/R23H270<IC<320320<270<IC<320320<D16K<120<120<120<120 S10P/D16K120<IC<170220<120<IC<170220<D16K/R23H270<IC<320320<270<IC<320320< D16A<120<120<120<120 S10P/D16A170<IC<220270<170<IC<220270< D16A/R23H270<IC<320320<270<IC<320320<W12 changes W12R<120<120<120<120S10P/W12R120<IC<170220≤120<IC<170220 ≤ W12R/R23H≤320320≤