Reproducing tna Operon Regulation in Vitro in an S-30 System

TRYPTOPHAN INDUCTION INHIBITS CLEAVAGE OF TnaC PEPTIDYL-tRNA*

Feng Gong and Charles YanofskyDagger

From the Department of Biological Sciences, Stanford University, Stanford, California 94305

Received for publication, September 28, 2000, and in revised form, October 19, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-tRNAPro. 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The enzyme tryptophanase catalyzes the degradation of L-tryptophan 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 (15-17).

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-tRNAPro. This finding implicates tryptophan-induced inhibition of cleavage of TnaC-peptidyl-tRNAPro as the event crucial to induction.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Plasmids-- A derivative of the E. coli A19 RNaseI- strain containing trpR Delta lacZ Delta trpEA2 tnaAbgl::Tn10, was constructed and used as the source of cell-free S-30 extracts. E. coli DH5 alpha  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 tnaA'-'trpE 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 tnaC'-'lacZ 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 PCR2 fragment (with an engineered HindIII site at its 5' end and a PstI site at its 3' end) containing the region from -195 to +6 (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 5' end and a PstI site at the 3' end) containing the region from -190 to +306 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 -160 upstream of tna promoter to a site just beyond the rpoBC terminator. We used two oligonucleotides, ECOR-160 (5'-ACGGAATTCCTGTTATTCCTCAACCC-3') and RPOC-ECOR (5'-ACGGAATTCCTTGCCGAGTTTGACTC-3'). EcoRI sites were introduced at both the 5' and 3' ends of this fragment. The fragment was digested with EcoRI and then circularized with T4 DNA ligase, resulting in CF-tna+306rpoBC"t". Using plasmid pGF1 as template and the same procedure, CF-tna+6rpoBC"t" was constructed. 2-3 µg of CF-tna+306rpoBC"t" or CF-tna+6rpoBC"t" were used in each 50-µl 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 µl) contains 35 mM Tris acetate, pH 8.0, 10 mM magnesium acetate, 200 mM potassium glutamate, 30 mM ammonium acetate, 2 mM dithiothreitol, 2 mM ATP, 0.5 mM each of CTP, UTP, and GTP, 20 mM phosphoenol pyruvate (trisodium salt), 0.25 mg/ml E. coli tRNA (Sigma), 0.3 unit/ml pyruvate kinase, 35 mg/ml polyethylene glycol (8000), 20 µg/ml folinic acid, 1 mM cyclic AMP, 200 µM each of 19 amino acids, with varying amounts of added L-tryptophan, and plasmid DNA or a circularized DNA fragment. All reactions were performed at 37 °C. To measure plasmid-directed beta -galactosidase and TnaA-TrpE synthesis, reaction mixtures were incubated for 40 min (except in time-course experiment). In protein labeling experiments, the unlabeled amino acid was replaced by 20 µCi of [35S]methionine, [3H]isoleucine, [3H]proline, [3H]tyrosine, or [3H]tryptophan (PerkinElmer Life Sciences, 1000 Ci/mmol). Details are presented in the legends. At the end of the incubation, a 5-µl 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 µCi of [alpha -33P]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-µl 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 µCi of [alpha -33P]UTP, 200 µM CTP, and 200 µg/ml rifampicin were added at the same time. Samples were taken at different intervals.

Identification of TnaC-peptidyl-tRNAPro-- After CF-tna+306rpoBC"t" directed cell-free transcription and translation (20 µCi of [35S]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 tRNAPro (tRNApro-Plus, 5'-CGGCACGTAGCGCAGCCTGGTAGC-3'; tRNApro-Minus, 5'-TGGTCGGCACGAGAGGATTTGAAC-3') were used. A S-30 reaction without the CF-tna+306rpoBC"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 µg of RNase A/ml or 50 µg of proteinase K/ml, mixed with an equal volume of 2× SDS gel loading buffer, and separated on a 10% Tricine-SDS protein gel.

Enzyme Assays-- beta -Galactosidase activity was determined as described (20).

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 µl of cell-free reaction mixture following a 20-min incubation at 37 °C. Fluorometer units were based on the following standard; 5 µl 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclic AMP-dependent Expression of a tnaC'-'lacZ 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 -35 region of the tna promoter (5, 10). Plasmid pPDG52 contains a tnaC'-'lacZ 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, beta -galactosidase activity was almost undetectable. By contrast, in the presence of cAMP and absence of added tryptophan, high levels of beta -galactosidase were produced. Addition of tryptophan increased beta -galactosidase production less than 2-fold. These results suggest that expression of tnaC'-'lacZ construct in this cell-free system is cAMP-dependent, and that tryptophan produced by protein turnover provides most of the tryptophan required for beta -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 beta -galactosidase monomer, which contains 39 Trp residues.



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Fig. 1.   Translational fusion constructs used in this study. A, schematic diagram of the tnaC'-'lacZ 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 tnaA'-'trpE 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.


                              
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Table I
Expression of tnaC'-'lacZ and tnaA'-'trpE gene fusions in vitro
In vitro S-30 reaction conditions and enzyme assay procedures are described under "Experimental Procedures." Each assay was performed in duplicate on at least three separate occasions, and the average is given here.

Basal and Induced Expression with the tnaA'-'trpE 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 tnaA'-'trpE 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 µM 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 [35S]methionine incorporation into the TnaA-TrpE protein, following SDS-polyacrylamide gel electrophoresis (Fig. 2). Template plasmid pGF4 contains a beta -lactamase gene, which also is expressed in vitro; beta -lactamase has four tryptophan residues, and therefore serves as an excellent internal reference to monitor the availability of charged tRNATrp for protein synthesis. In Fig. 2 it can be seen that there was no significant effect of tryptophan addition on beta -lactamase production. This suggests that there are appropriate levels of charged tRNATrp (tryptophan presumably generated by protein turnover) for synthesis of proteins like beta -lactamase, even in the absence of added tryptophan. A 25-fold increase in TnaA-TrpE synthesis was observed in the presence of 300 µM tryptophan; synthesis was assessed by counting the Cerenkov radiation of 35S-labeled TnaA-TrpE bands (Fig. 2, lane 5 versus lane 2) on the SDS-PAGE gels (data not shown). This increase agrees with the values in Table I, and suggests that the ASase assay results accurately reflect the levels of TnaA-TrpE protein produced in this S-30 system.



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Fig. 2.   A 10% Tricine-SDS-PAGE analysis of pGF4-directed in vitro translation products. In vitro reactions and Tricine-SDS-PAGE conditions are described under "Experimental Procedures." Polypeptide bands were labeled with [35S]methionine. The templates used were: no plasmid (lane 1), 5 nM pGF4 (lanes 2-5), 5 nM pBR322 (lane 6), and 15 nM pGF4 (lanes 7 and 8). Protein molecular weight standards (Low Range; Life Technologies, Inc.) were used as markers; their molecular weights are given. The positions of TnaA-TrpE and beta -lactamase are marked by arrows. *, 50 µM Trp was present in this reaction.

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 tnaA'-'trpE 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 tnaA'-'trpE fusion plasmid pGF4, and was not inducible by tryptophan (Table II). Addition of arginine had no effect on expression of pGF14 (TnaC Trp12 right-arrow 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.


                              
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Table II
Effect of added tryptophan and bicyclomycin on expression of tnaA'-'trpE plasmids
In vitro S-30 reaction conditions and the ASase assay procedure are described under "Experimental Procedures." Each assay was performed in duplicate on at least three separate occasions, and the average is given here.

Bicyclomycin Increases Basal Level Expression of All tnaA'-'trpE 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 µg/ml bicyclomycin was added in vitro with tnaA'-'trpE 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 time-course 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 µM 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 [35S]methionine-labeled TnaA-TrpE protein bands in lane 7 (without added tryptophan) and lane 8 (with 300 µM 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.



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Fig. 3.   Characterization of the cell-free S-30 system. In vitro protein synthesis and ASase assays were carried out as described under "Experimental Procedures." Induction ratios are shown in parentheses. A, a time-course experiment with plasmid pGF4-programmed TnaA-TrpE synthesis. B, effect of plasmid pGF4 concentration on TnaA-TrpE production, both in the presence and absence of added tryptophan. C, effect of DL-1-methyl-tryptophan (DL-1MT) concentration on the production of TnaA-TrpE protein.

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 tRNATrp 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 provided a 23-fold increase in ASase-specific activity over that observed in the absence of added 1MT. Increasing the tryptophan concentration from 50 µM to 300 µM (Fig. 2, lane 4 versus lane 5) also led to a significant increase in TnaA-TrpE production. Since tryptophanyl-tRNA synthase has a relatively high affinity for tryptophan (Km of 5 × 10-5 M; Ref. 26), the tRNATrp in our S-30 should be fully charged in the presence of 50 µM added tryptophan. Taken together, these findings suggest that tna induction is not based on the extent of charging of tRNATrp.

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 tnaA'-'trpE 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.


                              
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Table III
Addition of purified Rho lowers basal level expression when high concentrations of tnaA'-'trpE plasmids are used
In vitro S-30 reaction conditions and the ASase assay procedure are described under "Experimental Procedures." Each assay was performed in duplicate on at least three separate occasions, and the average is given here. Plasmid concentration is shown in parentheses.

300 µM Trp was added, as indicated.

ND, not determined.

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 µM 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 +6, CF-tna+6rpoBC"t". This circular template was used to direct the S-30 system (Fig. 4A). The predicted ~130-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.



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Fig. 4.   Demonstration of the requirement for cAMP for initiation of transcription of the tna operon. A, schematic representation of the circularized fragment (CF-tna+6rpoBC"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 ~130-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 µl each) were incubated at 37 °C for 10 min in the presence or absence of cyclic AMP and/or L-tryptophan. 20 µCi of [alpha -33P]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.

To confirm that the tryptophan induction observed with the tnaA'-'trpE translational fusion construct was due to relief from Rho-dependent termination, another circularized construct, CF-tna+306rpoBC"t", was examined (Fig. 5A). This construct contains the intact tna promoter, tna leader region to +306, followed by the rpoBC transcription terminator. Transcripts produced from this template that escape Rho-dependent termination should terminate at the rpoBC terminator, yielding a ~430-nucleotide read-through transcript (Fig. 5A). No transcripts were observed when cAMP or the template was omitted from the S-30 system (Fig. 5B, -cyclic 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, -Trp lane). Consistent with the translational results described above, no effect of tryptophan was observed when two templates with different tnaC mutations (ATG start codon changed to TAG, or Trp codon 12 changed to an Arg codon, in CF-tna+306rpoBC"t") were used (data not shown). Addition of bicyclomycin with cAMP increased basal level expression with all templates (data not shown).



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Fig. 5.   Requirements for in vitro synthesis of the read-through transcript. A, schematic representation of the circularized DNA fragment, CF-tna+306rpoBC"t", used as template to program the cell-free reactions in A. This fragment contains the intact tna promoter, the tna leader region to bp +306, followed by the rpoBC transcription terminator. The predicted tna transcripts in the absence or presence of tryptophan are also shown. B, analysis of the requirements for read-through transcription. A 6% polyacrylamide, 7 M urea gel was run with transcription/translation assay mixtures. S-30 reaction mixtures with 20 µCi of [alpha -33P]UTP (50 µl/reaction mixture) with the indicated additions were incubated at 37 °C for 20 min. The reactions were then stopped by phenol extraction, and the RNA samples were loaded on RNA gels. An arrow points to the position of the read-through transcript (RT).

Single-round Transcription Analyses in the S-30 System-- In the experiment described in Fig. 5B (-Trp lane), only low levels of terminated transcripts were observed. Why were bands of comparable intensity to those in the +Trp 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, [alpha -33P]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 Rho-mediated transcription termination, and degradation of these Rho-terminated transcripts. Comparison of the -Trp and +Trp 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 ~120-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").



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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-tna+306rpoBC"t" (see Fig. 5B) was used as template. S-30 reactions (100 µl) without CTP and UTP, in the absence or presence of tryptophan, were incubated at 37 °C for 10 min, then 50 µCi of [33P]UTP, 200 µM CTP, and 200 µg/ml rifampicin were added together to the reaction mixture. Samples (10 µl) 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-tna+306rpoBC"t" was used as template. Control reactions (+Trp) are shown on the left.

In other experiments, we tested templates analogous to CF-tna+306rpoBC"t" but bearing various tnaC mutations (ATG to TAG, or Trp12 right-arrow 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, -Trp lanes); thus, we do not show these results. We also examined the effects of adding the translation 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 tnaA'-'trpE 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-tna+306rpoBC"t", as template. This circularized template contains only the tna promoter, the tna leader region to bp +306, 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 [35S]methionine as label, two new bands (a ~3-kDa band and a ~25-kDa band) appear in the presence of added tryptophan. The ~3-kDa band (but not the ~25-kDa band) is present in the absence of added tryptophan. Neither of these bands is present when the CF-tna+306rpoBC"t" template is omitted from the S-30 reaction (Fig. 7A, lane 1). The ~3-kDa band could be TnaC, and the ~25-kDa band could be TnaC-peptidyl-tRNA. Some nonspecific products were also detected in all the reactions.



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Fig. 7.   Detecting TnaC peptide synthesis in vitro. CF-tna+306rpoBC"t" was used as template. A, [35S]methionine-labeled products produced in the presence and absence of added tryptophan. S-30 reaction mixtures (50 µl) were incubated at 37 °C for 20 min with 20 µCi of [35S]methionine; the reactions were stopped by acetone precipitation, boiled in Tricine-SDS sample buffer for 3 min, and then loaded on a 10% Tricine-SDS protein gel. A control sample, without template, is shown in lane 1. Arrows mark the positions of TnaC, TnaC-tRNAPro, and the nonspecific products. B, selective 3H labeling in the presence or absence of added tryptophan. S-30 reactions (50 µl) were incubated at 37 °C for 10 min (to decrease background labeling and let the transcription/translation reaction reach steady state), then [3H]proline, [3H]isoleucine, [3H]tyrosine, or [3H]tryptophan, was added. After 10 min of incubation, reactions were stopped by acetone precipitation, boiled in Tricine-SDS sample buffer for 3 min, and then loaded onto a 10% Tricine-SDS gel.

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). Accordingly, [3H]Trp, [3H]Ile, and [3H]Pro should label TnaC while [3H]Tyr should not. In vitro synthesis of the leader peptide was performed in the presence of [3H]Trp, [3H]Ile, [3H]Pro, or [3H]Tyr (Fig. 7B). As expected, when [3H]Tyr was used, no protein band was labeled, either in the absence or presence of tryptophan (lanes 5 and 6). The ~3-kDa peptide band was detected when [3H]Ile or [3H]Pro was used (lanes 1-4). [3H]Trp can label the ~3-kDa peptide in the absence of added unlabeled tryptophan (lane 7), and in the presence of 1MT as inducer (lane 8). No [3H]Trp-labeled band was visible when unlabeled tryptophan was added (lane 9). We also observed that, when a near-identical construct was used in which the tnaC start codon was replaced by TAG, no ~3-kDa or ~25-kDa product was detected (data not shown). Taken together, these data suggest that the ~3-kDa peptide band is TnaC. These findings also indicate that 1MT, an effective inducer, does not compete significantly with [3H]Trp incorporation into the TnaC peptide.

Identification of TnaC-peptidyl-tRNAPro-- The ~25-kDa band, observed only in the presence of added inducer (Fig. 7, A and B), has the same labeling characteristics as TnaC. Thus, [3H]Tyr did not label the ~25-kDa band (Fig. 7B, lane 6), whereas [3H]Pro (lane 2), [3H]Ile (lane 4), and [3H]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 ~25-kDa molecule cannot simply be a protein product resulting from translation of this read-through transcript. Thus, we considered it likely that the ~25-kDa band was a peptidyl-tRNA. To prove that this ~25-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 ~25-kDa band disappeared and the labeled product shifted to the TnaC position. The ~25-kDa molecule disappeared after proteinase K digestion (Fig. 8A). DNase treatment had no effect (data not shown). These data suggest that this ~25-kDa molecule contains TnaC linked to a RNA.



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Fig. 8.   Identification of TnaC-peptidyl-tRNAPro. A, a [35S]methionine-labeled ~25-kDa band was excised from a gel and treated with RNase A or proteinase K (for details, see "Experimental Procedures"), boiled in 1× Tricine-SDS sample buffer for 3 min, and then loaded onto a 10% Tricine-SDS protein gel. RNase A digestion shifts the ~25-kDa band to the TnaC band position. B, RT-PCR identification of tRNAPro. E. coli total tRNA (Sigma) was used as the control RT-PCR template (positive control, lane 1). The 25-kDa TnaC-tRNA band was excised, recovered from a long distance Tricine-SDS protein gel, and used as a RT-PCR template (lane 4). A S-30 reaction performed without the CF-tna+306rpoBC"t" template was also loaded onto the same gel, and the corresponding band was recovered as a control and used for RT-PCR (lane 2). A reaction mixture similar to the one used for lane 4, but without RT, was loaded in lane 3. A 10-bp DNA ladder (lane M, Life Technologies, Inc.) is shown.

The most likely identity of the ~25-kDa band is TnaC-peptidyl-tRNAPro (the C-terminal residue of TnaC is Pro). To examine this possibility, the ~25-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 tRNAPro. 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 ~25-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 ~25-kDa molecule observed in the presence of tryptophan is TnaC linked to tRNAPro.

One question raised by these results is whether the peptidyl-tRNA accumulated in the presence of tryptophan is only full-length TnaC-tRNAPro or whether other peptidyl-tRNAs are present in the peptidyl-tRNA band? Clearly TnaC-tRNAPro is present because the band is labeled by [3H]Pro and there is only a single Pro in TnaC (Fig. 7B). If the ~25-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 ~25-kDa band (data not shown). Taken together, our results indicate that the ~25-kDa species observed when tryptophan is present is TnaC-peptidyl-tRNAPro. In contrast to our findings with the template containing the wild type tnaC coding region, when we used template CF-tna+306rpoBC"t"bearing a tnaC mutation (Trp codon 12 changed to an Arg codon), no ~25-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-tRNAPro cleavage under inducing condition.

The Effect of Tryptophan Concentration on the Accumulation of TnaC, TnaC-tRNAPro, 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 experiments with our circularized CF-tna+306ropBC"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-tRNAPro signals were detected, with an apparent maximum at 1 mM L-tryptophan (Fig. 9A). When the read-through transcript level was determined, using [alpha -33P]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.



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Fig. 9.   Tryptophan dependence of TnaC, TnaC-tRNAPro, 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 µl each) were performed with CF-tna+306rpoBC"t" in the presence of the indicated concentrations of L-tryptophan. Incubation was at 37 °C for 10 min, then 20 µCi of [35S]methionine was added for 10 min, and the reactions were stopped by acetone precipitation, boiled in 1× Tricine-SDS sample buffer for 3 min, and then loaded onto a 10% Tricine-SDS gel. Levels of TnaC peptide and TnaC-tRNAPro were quantified using a PhosphorImager. The levels of TnaC and TnaC-tRNAPro 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-tan+306rpoBC"t" was used as template. The S-30 reaction mixtures (50 µl each), in the presence of the indicated concentrations of L-tryptophan, were incubated at 37 °C for 10 min, then 20 µCi of [33P]UTP and 200 µg/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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tryptophan-mediated induction of tna operon expression proceeds by preventing transcription termination at Rho factor-dependent 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-tRNAPro. 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-tRNAPro 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-tRNAPro is associated with the translating ribosome.

The S-30 Cell-free System-- E. coli strain RNaseI- trpR Delta lacZ Delta 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 NH3-dependent anthranilate synthase reaction that does not require TrpG-D protein (data not shown).

The Level of Tryptophanyl-tRNATrp-- 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 tRNATrp 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-tRNAPro (Fig. 7B), it now seems unlikely that the Trp residue at position 12 is modified. Similarly, induction appears to be independent of the level of tryptophanyl-tRNATrp, 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-tRNAPro 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 (22-24). 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-tRNAPro Synthesis, Ribosome Stalling at the tnaC Stop Codon, and the RNA Doublet-- The observation that TnaC-tRNAPro 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 5'-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 3' 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 +101 to +123, 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 reconstituted in an S-30-coupled transcription/translation system, with expression dependent on catabolite repression, Rho-dependent transcription termination, and tryptophan-induced antitermination. We also conclude that, in the presence of inducer, the newly synthesized TnaC-peptidyl-tRNAPro is resistant to cleavage. We assume that TnaC-peptidyl-tRNAPro 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 TnaC-peptidyl-tRNAPro is placed in the polypeptide exit tunnel of the 50 S ribosomal subunit; however, the presumed active segment of this TnaC-peptidyl-tRNAPro could interact with a surface of the peptide tunnel or with a region of the ribosomal A site (32, 33). Wherever TnaC-peptidyl-tRNAPro is located, its presence, plus inducing levels of tryptophan, appear to prevent release factor 2 from mediating cleavage of TnaC-peptidyl-tRNAPro. 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-tRNAPro, 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-tRNAPro 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).



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Fig. 10.   Model depicting the mechanism of tryptophan induction of tna operon expression. When a ribosome reaches the tnaC termination codon, UGA, in the presence of tryptophan, the 24-residue TnaC peptide (chain of circles) remains covalently joined to tRNAPro. Consequently the ribosome associated with the peptidyl-tRNA stalls at the tnaC stop codon. This blocks Rho's access to the BoxA and rut sites, and thereby prevents transcription termination. The positions at which amino acid residue changes have been shown to largely prevent induction are represented by black circles; positions where changes can result in constitutive expression are white circles; positions where changes result in mixed effects are indicated by white circles with a diagonal line; positions for which there are insufficient data are indicated as gray circles (14). The crucial Trp codon 12 is shown as an enlarged black circle. PTC, peptidyltransferase center.

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-tRNAPro prevented?


    ACKNOWLEDGEMENTS

We are indebted to Virginia Horn for constructing the E. coli strain used for S-30 preparations. We thank Peter von Hippel for the sample of purified E. coli Rho factor. We also thank Angela Valbuzzi, Kouacou Vincent Konan, and Ajith Kamath for critical reading of the manuscript. We thank the Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan, for providing the sample of bicyclomycin.


    FOOTNOTES

* This work was supported by Grant GM09738 (to C. Y.) from the United States Public Health Service.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 650-725-1835; Fax: 650-725-8221; E-mail: yanofsky@cmgm.stanford.edu.

Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M008892200

1 M. Eshoo and C. Yanofsky, unpublished data.

3 F. Gong and C. Yanofsky, unpublished results.


    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; RT, reverse transcription; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; 1MT, 1-methyltryptophan; ASase, anthranilate synthase; uORF, upstream open reading frame; bp, base pair(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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