Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

The enzyme tryptophanase catalyzes the degradation of Ltryptophan to indole, pyruvate, and ammonia. Degradation allows organisms to utilize tryptophan as a source of carbon, nitrogen, and energy (1,2). The tryptophanase reaction is reversible; thus, L-tryptophan can be formed from indole and L-serine, L-cysteine, or pyruvate and ammonia (3,4). The tna operon of Escherichia coli contains two major structural genes, tnaA, encoding tryptophanase, and tnaB, specifying a low affinity tryptophan permease (5,6). The tna operon promoter is separated from the tnaA structural gene by a 319-nucleotide leader region. This region encodes a 24-residue peptide, TnaC, that is essential for induction. Transcription of the tna operon is regulated by the combined action of catabolite repression and tryptophan-induced transcription antitermination. Regulation by catabolite repression requires the catabolite gene activator protein plus cyclic AMP, and is tryptophan-independent (5,7,8). Transcription termination/antitermination in the leader region of the operon is regulated in response to high levels of tryptophan. Studies in vivo and in vitro have shown that in the absence of tryptophan, transcription is subject to Rho-dependent termination at several transcription pause sites located between tnaC and tnaA (9,10). In the presence of inducing levels of tryptophan, termination at these sites is prevented (10). Translation of tnaC is essential for tryptophan induction (11). Specifically, inactivating the tnaC start codon, replacing the single Trp residue of TnaC with a different amino acid, or introducing several other amino acid changes in TnaC, prevents induction (13). 1 BoxA and rut sites located immediately adjacent to the tnaC stop codon are essential for Rho-dependent termination; alteration of either of these sites reduces termination (14). On the basis of these and other findings, it has been proposed that, in the presence of inducer, TnaC acts in cis on its associated translating ribosome to inhibit its release at the tnaC stop codon. Inhibition of ribosome release could block Rho's access to the BoxA or rut sites of the transcript and thereby prevent transcription termination (15)(16)(17).
To define the roles of TnaC, tryptophan, and Rho factor in regulation of tna operon expression, we developed a cell-free S-30 system in which regulation of tna operon could be studied. In this paper we show that the major regulatory features of tna operon observed in vivo can be duplicated in vitro. We examined transcription under inducing and noninducing conditions and demonstrate that in the absence of inducer transcription terminates at several sites in the leader region, and the terminated transcripts are then degraded. In the presence of tryptophan transcription pausing is observed, but the paused transcripts subsequently are elongated. We also detected and analyzed the synthesis and fate of the TnaC peptide. We show that tryptophan induction leads to the accumulation of TnaC-tRNA Pro . This finding implicates tryptophan-induced inhibition of cleavage of TnaC-peptidyl-tRNA Pro as the event crucial to induction.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids-A derivative of the E. coli A19 RNaseI Ϫ strain containing trpR ⌬lacZ ⌬trpEA2 tnaAbgl::Tn10, was constructed and used as the source of cell-free S-30 extracts. E. coli DH5 ␣ was occasionally employed for amplification of plasmids. All plasmid DNAs were purified by banding twice in cesium chloride-ethidium bromide gradients.
Plasmid pGF4 was constructed by inserting a HindIII-BamHI fragment containing a tnaAЈ-ЈtrpE translational fusion from pKG3 (constructed by Kirt Gish) into pBR322. pGF14 and pGF71 were constructed by replacing the HindIII-NsiI fragment of pGF4 containing the tna promoter through the ATG start codon of tnaA by an homologous fragment from pPDG14 and pPDG71 (constructed by P. Gollnick), respectively. In pPDG14, tnaC Trp codon 12 is replaced by an Arg codon; in pPDG71 the tnaC ATG start codon is replaced by TAG. Plasmid pPDG52 (constructed by P. Gollnick) contains a tnaCЈ-ЈlacZ translational fusion.
pGF24 was constructed by cloning the PstI-BamHI fragment containing E. coli rpoBC"t" from plasmid pSVS24 (constructed by V. Stewart) into pUC18. pGF1 was constructed by ligating a PCR 2 fragment (with an engineered HindIII site at its 5Ј end and a PstI site at its 3Ј end) containing the region from Ϫ195 to ϩ6 (relative to tna transcription initiation site) into pGF24 (HindIII and PstI sites). pGF25-00 was constructed by inserting a PCR fragment (with an engineered HindIII site at the 5Ј end and a PstI site at the 3Ј end) containing the region from Ϫ190 to ϩ306 into pGF24 (HindIII and PstI sites).
To facilitate clean transcription and translation, we prepared circular DNA templates. We performed PCR on plasmid pGF25-00 and amplified the region from Ϫ160 upstream of tna promoter to a site just beyond the rpoBC terminator. We used two oligonucleotides, ECOR-160 (5Ј-ACGGAATTCCTGTTATTCCTCAACCC-3Ј) and RPOC-ECOR (5Ј-ACGGAATTCCTTGCCGAGTTTGACTC-3Ј). EcoRI sites were introduced at both the 5Ј and 3Ј ends of this fragment. The fragment was digested with EcoRI and then circularized with T4 DNA ligase, resulting in CF-tnaϩ306rpoBC"t". Using plasmid pGF1 as template and the same procedure, CF-tnaϩ6rpoBC"t" was constructed. 2-3 g of CF-tnaϩ306rpoBC"t" or CF-tnaϩ6rpoBC"t" were used in each 50-l S-30 reaction.
In Vitro Protein Synthesis-S-30 extracts were prepared according to Zubay (18). Unless otherwise specified, the standard coupled transcription/translation reaction mixture (with a total volume of 50 l) contains 35  At the end of the incubation, a 5-l aliquot was removed, precipitated with five volumes of cold acetone, and pellets were analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) (19).
To measure RNA synthesis, unlabeled UTP was omitted from the incubation mixture. After 10 min of preincubation at 37°C, 20 Ci of [␣-33 P]UTP (PerkinElmer Life Sciences, 1000 -3000 Ci/mmol) was added to the reaction mixture, and samples were taken after another 10 min, and extracted with phenol. A 5-l aliquot was analyzed on a 6% polyacrylamide, 7 M urea gel. For single-round transcription (coupled with translation) assays, after 10 min of incubation at 37°C in the absence of added CTP and UTP, 50 Ci of [␣-33 P]UTP, 200 M CTP, and 200 g/ml rifampicin were added at the same time. Samples were taken at different intervals.
Identification of TnaC-peptidyl-tRNA Pro -After CF-tnaϩ306rpoBC"t" directed cell-free transcription and translation (20 Ci of [ 35 S]methionine, labeling for 20 min), reactions were stopped by acetone precipitation, centrifuged, and the pellets boiled in SDS gel loading buffer for 3 min. Samples were separated electrophoretically on a 10% Tricine-SDS-PAGE. The 25-kDa band was localized by autoradiography, excised, and recovered by immersion in a low pH buffer (20 mM Tris-HCl, pH 6.8, 50 mM NaCl) overnight at 4°C. The recovered 25-kDa molecules were used as RT-PCR template with an Access RT-PCR kit (Promega, Madison, WI). Two oligonucleotides specific for tRNA Pro (tRNApro-Plus, 5Ј-CGGCACGTAGCG-CAGCCTGGTAGC-3Ј; tRNApro-Minus, 5Ј-TGGTCGGCACGAGAGGATT-TGAAC-3Ј) were used. A S-30 reaction without the CF-tnaϩ306rpoBC"t" template was also loaded on the same gel, and the corresponding band was recovered as a negative control for RT-PCR.
For RNase digestion and proteinase K treatment, the recovered 25-kDa molecules were incubated for 10 min at 37°C with 10 g of RNase A/ml or 50 g of proteinase K/ml, mixed with an equal volume of 2ϫ SDS gel loading buffer, and separated on a 10% Tricine-SDS protein gel.
Anthranilate synthase activity was determined fluorometrically by measuring the conversion of chorismate plus glutamine to anthranilate (21). Specific activity was calculated as fluorometer units produced per 50 l of cell-free reaction mixture following a 20-min incubation at 37°C. Fluorometer units were based on the following standard; 5 l of a 1 mM solution of anthranilic acid were added to a tube containing the standard 0.5-ml reaction mixture, and the tube was incubated and processed with the assay tubes. Following acidification and extraction of anthranilate with ethyl acetate, the ethyl acetate extract from the standard tube was set at 100 fluorometer units. A blank reaction tube was treated similarly, and the ethyl acetate extract was set at zero. Fluorometer units were then determined for each assayed sample.
FIG. 1. Translational fusion constructs used in this study. A, schematic diagram of the tnaCЈ-ЈlacZ gene fusion fragment in plasmid pPDG52. In this construct, tnaC codon 20 is fused in frame to lacZ codon 8. B, schematic diagram of the tnaAЈ-ЈtrpE gene fusion fragment in plasmids pGF4, pGF14 and pGF71. This fragment contains the intact tna promoter, tna leader region, and the initiation codon ATG of tnaA in frame with the second codon of trpE. This cloning procedure substitutes histidine for glutamine at residue 2, yet functional TrpE is produced. In pGF14, the tnaC Trp12 codon, TGG, was replaced by an Arg codon, CGG. In pGF71, the tnaC start codon, ATG, was replaced by the TAG stop codon.

Cyclic AMP-dependent Expression of a tnaCЈ-ЈlacZ Fusion in an S-30
System-In previous studies it was shown that initiation of transcription of the tna operon is regulated by catabolite repression (8). A presumed cAMP-CAP complex binding site is located just upstream of the Ϫ35 region of the tna promoter (5,10). Plasmid pPDG52 contains a tnaCЈ-ЈlacZ translational fusion (Fig. 1A). This plasmid DNA was used as a reporter to examine catabolite repression-dependent expression of the tna operon in vitro. In this construct most of the tna leader region had been removed; thus, expression from the promoter could be tested independently of the effects of the tna operon downstream region and attenuation. The data in Table I show that, in the absence of cAMP and presence of added tryptophan, ␤-galactosidase activity was almost undetectable. By contrast, in the presence of cAMP and absence of added tryptophan, high levels of ␤-galactosidase were produced. Addition of tryptophan increased ␤-galactosidase production less than 2-fold. These results suggest that expression of tnaCЈ-ЈlacZ construct in this cell-free system is cAMP-dependent, and that tryptophan produced by protein turnover provides most of the tryptophan required for ␤-galactosidase synthesis. The less than 2-fold increase observed upon addition of tryptophan is probably not regulatory; it presumably is due to increased completion of synthesis of the 1024-residue ␤-galactosidase monomer, which contains 39 Trp residues.
Basal and Induced Expression with the tnaAЈ-ЈtrpE Construct-To study tryptophan induction of tna operon expression in vitro, we prepared a special construct, pGF4, containing the intact tna promoter-leader region followed by a translational fusion of the initial segment of tnaA fused to trpE of E. coli (Fig.  1B). The resulting fusion protein TnaA-TrpE would be tryptophan-free; thus, in vitro synthesis could be examined in the absence as well as the presence of tryptophan. The strain used to prepare the S-30 extract carried a trpR mutation and a trpE deletion; this extract has high levels of TrpG-D but no TrpE enzyme activity. As expected, expression of the tnaAЈ-ЈtrpE fusion was cAMP-dependent; without cAMP, very low levels of TnaA-TrpE enzyme activity were detected either in the absence or presence of tryptophan (Table I). In the presence of cAMP, addition of 300 M tryptophan led to a 17-fold increase in synthesis of the TnaA-TrpE protein. Addition of a tryptophan analog, 1MT, also led to a 20-fold increase (Table I). We believe that the increase in tryptophan-free TnaA-TrpE protein synthesis in the presence of tryptophan, or 1MT, is due to their action as inducers of antitermination in the leader region.
We also examined the effects of tryptophan addition on TnaA-TrpE synthesis by measuring [ 35 S]methionine incorporation into the TnaA-TrpE protein, following SDS-polyacrylamide gel electrophoresis (Fig. 2). Template plasmid pGF4 contains a ␤-lactamase gene, which also is expressed in vitro; ␤-lactamase has four tryptophan residues, and therefore serves as an excellent internal reference to monitor the availability of charged tRNA Trp for protein synthesis. In Fig. 2 it can be seen that there was no significant effect of tryptophan addition on ␤-lactamase production. This suggests that there are appropriate levels of charged tRNA Trp (tryptophan presumably generated by protein turnover) for synthesis of proteins like ␤-lactamase, even in the absence of added tryptophan. A 25-fold increase in TnaA-TrpE synthesis was observed in the presence of 300 M tryptophan; synthesis was assessed by counting the Cerenkov radiation of 35 Table I, and suggests that the ASase assay results accurately reflect the levels of TnaA-TrpE protein produced in this S-30 system.

Changing the tnaC Start Codon to TAG, or Replacing the tnaC Trp Codon 12 by an Arg Codon, Prevents Tryptophan
Induction-It was shown previously, in vivo, that synthesis of the intact TnaC peptide containing its crucial Trp residue is essential for tryptophan induction of tna operon expression (13). 1 To verify the role of tnaC translation in induction of the tna operon in the S-30 system, Trp codon 12 (TGG) and tnaC start codon (ATG) in the tnaAЈ-ЈtrpE construct were replaced by an Arg codon (CGG) and a stop codon (TAG), respectively. Replacing the tnaC start codon by a stop codon (pGF71, Fig.  1B) should eliminate leader peptide synthesis. Basal level expression by this plasmid was decreased 5-fold relative to that of the wild type plasmid pGF4, and induction by tryptophan was abolished (Table II). Replacing Trp codon 12 by an Arg codon (pGF14, Fig. 1B) should allow peptide synthesis to proceed to the normal TGA stop codon of tnaC, generating an altered peptide. TnaA-TrpE production directed by pGF14 was decreased 3-4-fold compared with that of the wild type tnaAЈ-ЈtrpE fusion plasmid pGF4, and was not inducible by trypto- The induction ratios observed upon addition of 300 M L-tryptophan (Trp) or DL-1-methytryptophan (1MT) are shown in parentheses.
phan (Table II). Addition of arginine had no effect on expression of pGF14 (TnaC Trp 12 3 Arg), confirming the in vivo finding that induction is not simply dependent on high concentrations of the amino acid encoded by codon 12 (13). These results suggest that translation of tnaC, and incorporation of Trp at codon position 12, are essential in establishing the basal level of tna operon expression, and for tryptophan induction, in vitro.

Bicyclomycin Increases Basal Level Expression of All tnaAЈ-ЈtrpE Constructs-Bicyclomycin is a specific inhibitor of E. coli
Rho factor. This antibiotic inhibits the poly(C)-stimulated ATPase activity of E. coli Rho factor (22,23). Consistent with this finding, it has been shown that bicyclomycin increases basal level expression of the tna operon in vivo (24). Addition of an appropriate concentration of this antibiotic directly to the S-30 system should therefore inhibit Rho factor activity and increase basal level expression of the tna operon. To examine this possibility, 50 g/ml bicyclomycin was added in vitro with tnaAЈ-ЈtrpE plasmids that did or did not have tnaC mutations. Bicyclomycin relieved Rho-dependent termination in the tna operon, irrespective of whether an intact TnaC peptide could be synthesized (Table II).
Characterization of tna Operon Regulation in the S-30 System-To optimize reaction conditions, we examined three variables: reaction time, plasmid pGF4 concentration, and the concentration of inducer. Fig. 3A presents the results of a timecourse experiment performed in the presence of inducer. TnaA-TrpE enzyme activity was observed to increase linearly for 40 min, whereupon it began to level off. In the absence of inducer, there was no significant increase in TnaA-TrpE activity. Plasmid concentration was varied, as shown in Fig. 3B. When 5 nM pGF4 was present, there was 18-fold induction by 300 M tryptophan. Increasing the pGF4 plasmid concentration to 10 nM elevated basal level expression appreciably, and there was only 1.2-fold induction by added tryptophan. When the pGF4 plasmid concentration was increased to 15 nM or greater, basal level expression increased further and there was no significant effect of added tryptophan. These results are consistent with the intensities of the TnaA-TrpE bands observed on a SDS-PAGE gel (Fig. 2, compare lanes 7 and 8); when 15 nM pGF4 was used, the [ 35 S]methionine-labeled TnaA-TrpE protein bands in lane 7 (without added tryptophan) and lane 8 (with 300 M tryptophan) were indistinguishable. These data suggest that the S-30 system employed is limiting for some component required for efficient transcription termination under noninducing condition.
1MT, an analog of tryptophan, effectively induces tna operon expression in vivo (25). It is thought that 1MT induces tna operon without being charged onto tRNA Trp or being incorporated into protein (see below) (13). The effect of varying 1MT concentration on TnaA-TrpE production in our S-30 system is shown in Fig. 3C. A concentration of 0.5-1 mM was sufficient to fully induce tna operon expression; maximum induction pro-   Rho Is Limiting in Our Cell-free System-One unexpected finding was that, when the concentration of plasmid pGF4 was increased from 5 to 15 nM, basal level expression of tnaAЈ-ЈtrpE increased dramatically (Fig. 3B). This observation suggests that a factor(s) responsible for (or participating in) basal level expression in the S-30 system might be limiting, or titrated out. To determine whether increased translation of tnaC is responsible for this presumed titration effect, constructs with mutations in tnaC that prevent induction were tested. It can be seen in Table III that, with 15 nM pGF71 (or pGF14) (Fig. 1B) as template, the results obtained are similar to those observed with pGF4 (Fig. 3B). These findings indicate that translation of tnaC is not responsible for the apparent titration effect.
A second reasonable explanation is that Rho may become limiting because the additional tna transcripts produced contribute BoxA and rut binding sites that sequester much of the available Rho. If this is the case, addition of purified Rho protein should reverse the titration effect. As expected, when 6 M Rho was added to the reaction mixture (Table III), both the basal and induced levels of tna operon expression were lowered, and now 6-fold tryptophan induction was observed with wild type tnaC plasmid pGF4. No induction was observed with either pGF14 or pGF71, as expected. These findings support the interpretation that Rho is responsible for setting the basal level of expression of the tna operon.
Measurement of tna mRNA Synthesis-To demonstrate directly that tryptophan has no effect on cAMP-dependent transcription initiation at the tna promoter, a circularized DNA template was constructed that contains the intact tna promoter but lacks the leader region beyond bp ϩ6, CF-tnaϩ6rpoBC"t". This circular template was used to direct the S-30 system (Fig.  4A). The predicted ϳ130-nucleotide transcript would contain only the first 6 nucleotides of the tna leader region, immediately followed by the E. coli rpoBC terminator (rpoBC"t"). In Fig. 4B it can be seen that with this template transcription initiation from the tna promoter was highly efficient and was dependent on the presence of cyclic AMP in the reaction mixture. No effect of added L-tryptophan was evident.
To confirm that the tryptophan induction observed with the tnaAЈ-ЈtrpE translational fusion construct was due to relief from Rho-dependent termination, another circularized construct, CF-tnaϩ306rpoBC"t", was examined (Fig. 5A). This construct contains the intact tna promoter, tna leader region to ϩ306, followed by the rpoBC transcription terminator. Transcripts produced from this template that escape Rho-dependent termination should terminate at the rpoBC terminator, yielding a ϳ430-nucleotide read-through transcript (Fig. 5A). No transcripts were observed when cAMP or the template was omitted from the S-30 system (Fig. 5B, Ϫcyclic AMP lane). In the presence of cAMP and added tryptophan, a prominent read-through (RT) transcript band was observed (Fig. 5B, last  lane). Only a faint read-through transcript was produced when tryptophan was omitted (Fig. 5B, ϪTrp lane). Consistent with the translational results described above, no effect of tryptophan was observed when two templates with different tnaC FIG. 4. Demonstration of the requirement for cAMP for initiation of transcription of the tna operon. A, schematic representation of the circularized fragment (CF-tnaϩ6rpoBC"t") used as template to program the cell-free reactions illustrated in A. The first 6 nucleotides of the tna leader transcript were followed by the E. coli rpoBC terminator (rpoBC"t"). The source of the predicted ϳ130-nucleotide transcript is shown. B, analysis of transcription in an S-30 system. A 6% polyacrylamide, 7 M urea gel was used. Transcription initiation at the tna promoter was cAMP-dependent and tryptophan-independent. The S-30 reactions (50 l each) were incubated at 37°C for 10 min in the presence or absence of cyclic AMP and/or L-tryptophan. 20 Ci of [␣-33 P]UTP was then added, and after a 5-min incubation, the reactions were stopped by phenol extraction and the contents loaded onto an RNA gel. mutations (ATG start codon changed to TAG, or Trp codon 12 changed to an Arg codon, in CF-tnaϩ306rpoBC"t") were used (data not shown). Addition of bicyclomycin with cAMP increased basal level expression with all templates (data not shown).

Single-round Transcription Analyses in the S-30
System-In the experiment described in Fig. 5B (ϪTrp lane), only low levels of terminated transcripts were observed. Why were bands of comparable intensity to those in the ϩTrp lane not also seen? In an attempt to explain this observation, we analyzed transcription more closely using the "single-round transcription" approach (27). Using this procedure, template is incubated in the S-30 extract plus or minus tryptophan for 10 min at 37°C without added CTP or UTP, and then rifampicin, [␣-33 P]UTP, and CTP are added. Samples are removed at various intervals thereafter and examined by urea gel electrophoresis. In the absence of added tryptophan (Fig. 6A), multiple paused transcripts are visible at 1 min; these bands subsequently disappear, presumably as a result of RNA degradation. In the presence of added tryptophan (Fig. 6A), comparable bands are observed at 1 min, but thereafter distinct intermediate length bands appear and the read-through band becomes more prominent. When the identical experiment is performed in the presence of bicyclomycin (Fig. 6B), the mRNA banding pattern in the absence of added tryptophan more closely resembles the pattern observed in the presence of tryptophan. It would appear, therefore, that the differences observed in the absence of bicyclomycin are the result of Rhomediated transcription termination, and degradation of these Rho-terminated transcripts. Comparison of the ϪTrp and ϩTrp lanes (Fig. 6A) suggests that pausing may occur at most of the same sites under both conditions. One exception to this generalization is evident; in the presence of added tryptophan, a ϳ120-nucleotide RNA doublet can be seen near the bottom of the gel. We believe that this doublet is produced by degradation of the ribosome-bearing read-through transcript that is produced in the presence of tryptophan (see "Discussion").
In other experiments, we tested templates analogous to CF-tnaϩ306rpoBC"t" but bearing various tnaC mutations (ATG to TAG, or Trp 12 3 Arg). Both in the presence and absence of tryptophan, all templates gave RNA expression patterns similar to those obtained with the wild type template in the absence of tryptophan (Fig. 6A, ϪTrp lanes); thus, we do not show these results. We also examined the effects of adding the translation

FIG. 6. Single-round transcription analyses examining the effects of added tryptophan and bicyclomycin.
A, single-round transcription analyses (coupled with translation) were performed in an S-30 system. Circularized fragment CF-tnaϩ306rpoBC"t" (see Fig. 5B) was used as template. S-30 reactions (100 l) without CTP and UTP, in the absence or presence of tryptophan, were incubated at 37°C for 10 min, then 50 Ci of [ 33 P]UTP, 200 M CTP, and 200 g/ml rifampicin were added together to the reaction mixture. Samples (10 l) were taken at indicated time points, stopped by phenol extraction, and loaded on an RNA gel. The read-through transcript (RT) and the RNA doublet observed only in the presence of tryptophan are marked by arrows. B, effect of addition of the Rho inhibitor bicyclomycin, to a reaction mixture incubated in the absence of tryptophan. Single-round transcription assays (coupled to translation) were performed as described in A. CF-tnaϩ306rpoBC"t" was used as template. Control reactions (ϩTrp) are shown on the left. inhibitors streptomycin, kasugamycin, or chloramphenicol. Addition of any of these drugs eliminated the tryptophan induction effect (data not shown). These results confirm the importance of tnaC translation in induction of tna operon; tnaC translation is addressed in the next section.
Synthesis of the TnaC Peptide in Vitro-Previous attempts to synthesize TnaC of E. coli in an S-30 system were unsuccessful (28). When we used the tnaAЈ-ЈtrpE construct pGF4 to program protein synthesis, we also did not observe TnaC peptide production. Synthesis was examined using a Tricine-SDS protein gel (see Fig. 2). To improve our ability to detect TnaC, we decided to eliminate competition for transcription or translation. Therefore, we changed our strategy and used a circularized small PCR fragment, CF-tnaϩ306rpoBC"t", as template. This circularized template contains only the tna promoter, the tna leader region to bp ϩ306, followed by the rpoBC terminator. The only transcripts expected would be those initiated at the tna promoter, and the resulting peptide product would be TnaC. In Fig. 7A it can be seen that, with [ 35 S]methionine as label, two new bands (a ϳ3-kDa band and a ϳ25-kDa band) appear in the presence of added tryptophan. The ϳ3-kDa band (but not the ϳ25-kDa band) is present in the absence of added tryptophan. Neither of these bands is present when the CF-tnaϩ306rpoBC"t" template is omitted from the S-30 reaction (Fig. 7A, lane 1). The ϳ3-kDa band could be TnaC, and the ϳ25-kDa band could be TnaC-peptidyl-tRNA. Some nonspecific products were also detected in all the reactions. The 2.9-kDa 24-residue TnaC peptide predicted from the tnaC nucleotide sequence should contain 1 Trp, 4 Ile, 1 Pro, and no Tyr residues (5 1-4).  9). We also observed that, when a near-identical construct was used in which the tnaC start codon was replaced by TAG, no ϳ3-kDa or ϳ25-kDa product was detected (data not shown). Taken together, these data suggest that the ϳ3-kDa peptide band is TnaC. These findings also indicate that 1MT, an effective inducer, does not compete significantly with [ 3 H]Trp incorporation into the TnaC peptide.
Identification of TnaC-peptidyl-tRNA Pro -The ϳ25-kDa band, observed only in the presence of added inducer (Fig. 7, A  and B), has the same labeling characteristics as TnaC. Thus, [ 3 H]Tyr did not label the ϳ25-kDa band (Fig. 7B, lane 6

), whereas [ 3 H]Pro (lane 2), [ 3 H]Ile (lane 4), and [ 3 H]Trp (lane 8)
did. Since the read-through transcript produced from our template should be about 430 nucleotides in length (Fig. 5, A and  B), this ϳ25-kDa molecule cannot simply be a protein product resulting from translation of this read-through transcript. Thus, we considered it likely that the ϳ25-kDa band was a peptidyl-tRNA. To prove that this ϳ25-kDa molecule is indeed TnaC linked to another molecule, and to further characterize this molecule, the 25-kDa band was excised from the gel and treated with RNase A, proteinase K (Fig. 8A), and DNase. After RNase A treatment, the ϳ25-kDa band disappeared and the labeled product shifted to the TnaC position. The ϳ25-kDa molecule disappeared after proteinase K digestion (Fig. 8A). DNase treatment had no effect (data not shown). These data suggest that this ϳ25-kDa molecule contains TnaC linked to a RNA.
The most likely identity of the ϳ25-kDa band is TnaC-peptidyl-tRNA Pro (the C-terminal residue of TnaC is Pro). To examine this possibility, the ϳ25-kDa molecule was separated from E. coli total tRNA by long distance Tricine-SDS-PAGE, located by autoradiography, excised, and used as template for RT-PCR using primers based on the sequence of tRNA Pro . A S-30 sample that had not been incubated with the circularized DNA template was also loaded on the same gel, and the corresponding band was recovered as a control to test for tRNA contamination. The S-30 control did not yield a product (Fig.  8B, lane 2), and the lane in which read-through was omitted also lacked a product (lane 3), while a 70 -80 bp PCR product was observed using the ϳ25-kDa molecule as template (lane 4). As a positive control, total tRNA from E. coli (Sigma) was used as template. We observed the same 70 -80-bp product plus a shorter product (lane 1). These findings indicate that the ϳ25-kDa molecule observed in the presence of tryptophan is TnaC linked to tRNA Pro .
One question raised by these results is whether the peptidyl-tRNA accumulated in the presence of tryptophan is only fulllength TnaC-tRNA Pro or whether other peptidyl-tRNAs are present in the peptidyl-tRNA band? Clearly TnaC-tRNA Pro is present because the band is labeled by [ 3 H]Pro and there is only a single Pro in TnaC (Fig. 7B). If the ϳ25-kDa band is a mixture of peptidyl-tRNAs, we might expect to see a ladder or smeared band on SDS gels. To the contrary, we ran long 10% and 15% SDS gels and always observed only a sharp ϳ25-kDa band (data not shown). Taken together, our results indicate that the ϳ25-kDa species observed when tryptophan is present is TnaC-peptidyl-tRNA Pro . In contrast to our findings with the template containing the wild type tnaC coding region, when we used template CF-tnaϩ306rpoBC"t"bearing a tnaC mutation (Trp codon 12 changed to an Arg codon), no ϳ25-kDa TnaC(W12R)-tRNA accumulation was observed (data not shown). This finding suggests that Trp at TnaC position 12 is required for the inhibition of TnaC-peptidyl-tRNA Pro cleavage under inducing condition.
The Effect of Tryptophan Concentration on the Accumulation of TnaC, TnaC-tRNA Pro , and the Read-through Transcript-As shown above, tryptophan, the inducer of the tna operon, plays a crucial role in the accumulation of peptidyl-tRNA and the production of the read-through transcript. To determine the tryptophan concentration dependence of the events leading to peptidyl-tRNA and read-through transcript production, we performed experi-ments with our circularized CF-tnaϩ306ropBC"t" DNA template, and varied the tryptophan concentration (Fig. 9). As the tryptophan concentration in the S-30 reaction was increased, stronger TnaC peptide and TnaC-tRNA Pro signals were detected, with an apparent maximum at 1 mM L-tryptophan (Fig. 9A). When the readthrough transcript level was determined, using [␣-33 P]UTP to label transcripts in the presence of increasing concentrations of tryptophan, the maximum level of read-through transcript was observed at the highest tryptophan concentration tested, 0.5 mM (Fig. 9B). These findings indicate that, in the S-30 system, a tryptophan concentration in excess of 0.25 mM is probably required to obtain 50% or greater induction of tna operon expression. DISCUSSION Tryptophan-mediated induction of tna operon expression proceeds by preventing transcription termination at Rho factordependent termination sites in the leader region of the operon (9 -11). The simplest model consistent with all of our previous experimental findings is that, in the presence of inducing levels of tryptophan, the nascent TnaC peptide acts in cis on its translating ribosome to inhibit its release at the tnaC stop codon. The stalled ribosome would presumably block Rho's access to the BoxA and rut site adjacent to the tnaC stop codon, and thereby prevent Rho-mediated transcription termination (29).
In this study we successfully reproduced all the in vivo features of tna operon regulation by tryptophan-induced transcription antitermination using a coupled transcription/translation S-30 system from E. coli. In addition, we provide the first demonstration of E. coli TnaC peptide synthesis in the S-30 system (in vitro synthesis of Proteus vulgaris TnaC has been shown (Ref. 28)). Most importantly, we show that the presence of inducing levels of tryptophan leads to the accumulation of TnaC-tRNA Pro . We also show that moderately high tryptophan concentrations are required for induction and for peptidyl-tRNA accumulation. Our results provide direct support for the hypothesis that, in the presence of inducing levels of tryptophan, TnaC-tRNA Pro is not cleaved. This peptidyl-tRNA presumably blocks release of the translating ribosome at the tnaC stop codon, and prevents Rho action. Our preliminary findings (data not shown) demonstrate that the peptidyl-tRNA Pro is associated with the translating ribosome.
The S-30 Cell-free System-E. coli strain RNaseI Ϫ trpR ⌬lacZ ⌬trpEA2 tnaA bgl::Tn10 was used to prepare our S-30 extracts. This strain was derived from the classic RNase I minus A-19 strain. trpE and lacZ deletions were introduced into this strain; consequently, any TrpE or LacZ activity detected in the S-30 extract would result from plasmid-programmed synthesis. This strain is trp repressor minus; thus, S-30 extracts from this strain have elevated levels of the TrpG-D protein (30) that are sufficient to fully activate all the TnaA-trpE protein synthesized during the course of an experiment. This conclusion has been confirmed by measuring TnaA-TrpE activity in the NH 3 -dependent anthranilate synthase reaction that does not require TrpG-D protein (data not shown).
The Level of Tryptophanyl-tRNA Trp -How added tryptophan serves as the signal that leads to inhibition of Rho-dependent termination is a basic unanswered question. Translation of Trp codon 12 of tnaC by tRNA Trp is believed to be essential for tryptophan-induced antitermination with the wild type tna operon (13). It has been suggested that the TnaC peptide might be modified in some manner when excess tryptophan is present (15). Since full induction of the tna operon in the S-30 system is also observed with 1 mM 1MT as inducer, and since unlabeled 1MT does not appear to reduce incorporation of labeled tryptophan into TnaC or TnaC-tRNA Pro (Fig. 7B), it now seems unlikely that the Trp residue at position 12 is modified. Simi- larly, induction appears to be independent of the level of tryptophanyl-tRNA Trp , since we find, in agreement with in vivo data, that the tryptophan concentration required for appreciable tna operon induction is higher than for general protein synthesis. This is logical for an amino acid catabolic enzyme, since the degradative operon should only be expressed when its substrate is well in excess of the level required for tRNA charging and protein synthesis. It seems likely, therefore, that there is a specific binding site for tryptophan in the translating ribosome, and that tryptophan must be at this site for induction to occur. Whether TnaC-tRNA Pro contributes to this binding site is a question that requires experimental attention. In any event TnaC peptide synthesized in the presence and absence of inducer should be sequenced to be certain that induction does not involve a modification of one of the residues in the TnaC peptide. This is now feasible because we can produce the TnaC peptide in vitro.
Rho-dependent Termination-Rho action is largely responsible for the low level of expression observed in vivo in the absence of inducer. Rho-dependent transcription termination at sites located in the tna leader region has been characterized previously, both in vivo and in an in vitro purified transcription system (9). Bicyclomycin inhibition of termination (Table II and Fig. 6B) and Rho titration (Table III) are consistent with this conclusion. The antibiotic bicyclomycin is known to interact with Rho and inhibit its action (22)(23)(24). Addition of bicyclomycin to our in vitro system led to a 15-fold increase in basal level expression (Table II). In a recent study, it has been shown that a BoxA site and a rut site (both are adjacent to the tnaC stop codon) are crucial for Rho action in vivo (14).
TnaC-tRNA Pro Synthesis, Ribosome Stalling at the tnaC Stop Codon, and the RNA Doublet-The observation that TnaC-tRNA Pro accumulates in the presence of inducing levels of tryptophan suggests that the translating ribosome may be stalled at the tnaC stop codon. If this is correct, the associated transcript should not be available for subsequent rounds of translation. In the absence of added tryptophan, TnaC would presumably be synthesized and released. Thus, one might expect to detect a higher level of TnaC peptide in the absence versus the presence of added tryptophan. However, in the presence of tryptophan, TnaC would exist as two species, free and as peptidyl-tRNA. Unfortunately, the TnaC peptide is labile in the S-30 system (data not shown); therefore, we could not reliably measure the level of TnaC that is synthesized.
Appearance of the RNA doublet only in the presence of tryptophan provides additional support for the interpretation that induction results in ribosome stalling at the tnaC stop codon. It is very likely that the RNA doublet arises from RNA processing. When we employ a template with a 5-bp deletion at the 5Ј-end of the tna leader region instead of the wild type template, a corresponding decrease is observed in the length of RNA doublet (data not shown). This establishes that the 3Ј end of the doublet is identical using either template. The normal length RNA doublet was also observed when a template was used that has the rut region deleted (from bp ϩ101 to ϩ123, just beyond the stop codon of tnaC), suggesting that the RNA secondary structure that forms just beyond the tnaC stop codon (9) is not responsible for formation of the doublet (data not shown). When streptolydigin was added to an incubation mixture to inhibit all ongoing transcription, RNA doublet accumulation was still observed, consistent with doublet formation resulting from RNA processing.
Our combined results show that tna operon regulation can be FIG. 9. Tryptophan dependence of TnaC, TnaC-tRNA Pro , and read-through transcript production. A, a 10% Tricine-SDS protein gel of S-30 reactions performed at increasing tryptophan concentrations. S-30 reactions (50 l each) were performed with CF-tnaϩ306rpoBC"t" in the presence of the indicated concentrations of L-tryptophan. Incubation was at 37°C for 10 min, then 20 Ci of [ 35 S]methionine was added for 10 min, and the reactions were stopped by acetone precipitation, boiled in 1ϫ Tricine-SDS sample buffer for 3 min, and then loaded onto a 10% Tricine-SDS gel. Levels of TnaC peptide and TnaC-tRNA Pro were quantified using a PhosphorImager. The levels of TnaC and TnaC-tRNA Pro in the 1 mM tryptophan lane were set at 100%, respectively. B, a 6% polyacrylamide, 7 M urea gel of a transcription/translation reaction examining the effect of increasing tryptophan concentration on the production of read-through (RT) transcript. CF-tanϩ306rpoBC"t" was used as template. The S-30 reaction mixtures (50 l each), in the presence of the indicated concentrations of L-tryptophan, were incubated at 37°C for 10 min, then 20 Ci of [ 33 P]UTP and 200 g/ml rifampicin were added. After 1, 5, or 15 min of incubation, samples were taken and reactions were stopped by phenol extraction and loaded on an RNA gel.  (14). The crucial Trp codon 12 is shown as an enlarged black circle. PTC, peptidyltransferase center. reconstituted in an S-30-coupled transcription/translation system, with expression dependent on catabolite repression, Rhodependent transcription termination, and tryptophan-induced antitermination. We also conclude that, in the presence of inducer, the newly synthesized TnaC-peptidyl-tRNA Pro is resistant to cleavage. We assume that TnaC-peptidyl-tRNA Pro is in the P site of the translating ribosome, and that the tnaC UGA stop codon is in the ribosomal A site. We previously reported that inactivation or overproduction of release factor 3 affects both basal level expression and induction of the tna operon (31). These findings imply that the release factor 3-mediated event in normal ribosome release can influence the unusual events associated with induction. A schematic representation of the hypothetical stalled translation termination complex that forms in the presence of tryptophan is shown in Fig. 10. In this representation the peptidyl portion of TnaCpeptidyl-tRNA Pro is placed in the polypeptide exit tunnel of the 50 S ribosomal subunit; however, the presumed active segment of this TnaC-peptidyl-tRNA Pro could interact with a surface of the peptide tunnel or with a region of the ribosomal A site (32,33). Wherever TnaC-peptidyl-tRNA Pro is located, its presence, plus inducing levels of tryptophan, appear to prevent release factor 2 from mediating cleavage of TnaC-peptidyl-tRNA Pro . Similar findings have been described by Cao and Geballe (34 -36) in studies on translational regulation of gene expression in the cytomegalovirus. They have shown that translation of an upstream open reading frame, uORF2, regulates translation of a downstream coding region, corresponding to gene UL4. The uORF2 polypeptide, 22 residues in length, like TnaC, accumulates as a peptidyl-tRNA Pro , at the stop codon of the uORF coding region. This peptidyl-tRNA also is resistant to cleavage, resulting in ribosome stalling and blockage of translation of the downstream coding region for UL4 (35). The features of this example also suggest that certain amino acid sequences in a nascent peptide can interfere with peptidyl-tRNA cleavage when the translating ribosome is reading a stop codon. Addition of puromycin does not result in cleavage of this peptidyl-tRNA, and its release from the ribosome (37). By contrast, TnaC-peptidyl-tRNA Pro can be released and cleaved in response to puromycin. 3 Somewhat related examples in bacteria concern antibiotic inhibition of peptide chain elongation, leading to ribosome stalling. This influences the availability of a downstream nucleotide sequence needed for translation initiation. Chloramphenicol action during translational attenuation in the CAT operon is perhaps the best understood example of this type (12).
Four important questions remain unanswered regarding tna operon regulation: how is tryptophan recognized, what is the role of TnaC, what is the role of Trp at position 12, and, under inducing conditions, how is cleavage of TnaC-tRNA Pro prevented?