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Journal of Bacteriology, March 2006, p. 1892-1898, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1892-1898.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA2Pro Availability

Ming Gong, Feng Gong,{dagger} and Charles Yanofsky*

Department of Biological Sciences, Stanford University, Stanford, California

Received 31 October 2005/ Accepted 9 December 2005


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ABSTRACT
 
Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction results from ribosome stalling after translation of tnaC, the coding region for a 24-residue leader peptide. The last sense codon of tnaC, proline codon 24 (CCU), is translated by tRNA2Pro. We analyzed the consequences of overexpression of tnaC from a multicopy plasmid and observed that under inducing conditions more than 60% of the tRNA2Pro in the cell was sequestered in ribosomes as TnaC-tRNA2Pro. The half-life of this TnaC-tRNA2Pro was shown to be 10 to 15 min under these conditions. Plasmid-mediated overexpression of tnaC, under inducing conditions, reduced cell growth rate appreciably. Increasing the tRNA2Pro level relieved this growth inhibition, suggesting that depletion of this tRNA was primarily responsible for the growth rate reduction. Growth inhibition was not relieved by overexpression of tRNA1Pro, a tRNAPro that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA1Pro, also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA1Pro. These findings establish that the growth inhibition caused by tnaC overexpression during induction by tryptophan is primarily a consequence of tRNAPro depletion, resulting from TnaC-tRNAPro retention within stalled, translating ribosomes.


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INTRODUCTION
 
Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction requires attempted translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase (4, 19). Both Trp12 and Pro24 of TnaC are essential for induction (6, 15, 10). The key feature of this antitermination mechanism has been shown to be retention of uncleaved TnaC-tRNA2Pro within the translating ribosome (9). The translating ribosome stalls at the tnaC stop codon (UGA) because TnaC-tRNA2Pro resists cleavage, and this stalling blocks Rho factor's access to the rut site in the tna transcript (11), thereby preventing transcription termination in the leader region of the operon.

During normal protein synthesis each ribosome translating an open reading frame continues translation until it reaches a termination codon. Then, a release factor (RF) promotes hydrolysis of the peptidyl-tRNA, releasing the polypeptide product and its previously associated tRNA (2, 13). In some instances, a peptidyl-tRNA dissociates from the ribosome before hydrolysis of the ester bond (3, 12, 16). This "drop-off" peptidyl-tRNA is believed to be hydrolyzed by the enzyme peptidyl-tRNA hydrolase (Pth), allowing the tRNA to be recycled for continued protein synthesis. Pth has been shown to be essential in E. coli (1, 18), presumably because efficient tRNA recycling is necessary for continued protein synthesis and cell growth. For example, it has been shown that minigene overexpression in E. coli can inhibit both translation and cell growth (14, 20). Inhibition occurs because cells fail to cleave the peptidyl-tRNA released from ribosomes efficiently, resulting in depletion of essential tRNA species (14, 20).

In an earlier study it was observed that overexpression of the tnaC operon leader region from a multicopy plasmid inhibited expression of the chromosomal tna operon (6). The cause of this inhibition was not established. Subsequent in vitro studies demonstrated that the normal mechanism of tryptophan induction of tna operon expression involves inhibition of RF2-mediated peptidyltransferase cleavage of TnaC-tRNA2Pro, resulting in retention of this peptidyl-tRNA within the translating ribosome (8). If this inhibition of TnaC-tRNA2Pro cleavage occurred in vivo it might deplete the cell of sufficient free tRNA2Pro to continue normal protein synthesis. This depletion could explain why overexpression of tnaC results in reduced expression of the chromosomal tna operon (6).

To determine whether tryptophan induction does lead to TnaC-tRNA2Pro accumulation in vivo, tnaC of E. coli was overexpressed from its own promoter by using a multicopy plasmid-based system. Accumulation of TnaC-tRNA2Pro in vivo was observed, and this TnaC-tRNA2Pro was shown to be associated with ribosomes. Cell growth rate was reduced when tryptophan was present, but only when cells contained multiple copies of the wild-type tnaC, not a nonfunctional tnaC, W12R tnaC, in which Trp12 of TnaC was replaced by Arg. Approximately 60% of the tRNA2Pro in tryptophan-induced cells was found to be retained as TnaC-tRNA2Pro; this TnaC-tRNA2Pro was within the ribosomal fraction. Overexpression of tRNA2Pro relieved this growth rate reduction. Substituting Pro codon CCG, read by tRNA1Pro, for the wild-type tnaC Pro24 codon, CCU, read by tRNA2Pro, also resulted in growth rate reduction; this reduction was relieved by overexpressing tRNA1Pro. These findings establish that tryptophan-induced sequestration of tRNAPro as TnaC-tRNAPro within ribosomes stalled during tnaC translation is the cause of the observed growth rate reduction.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. All strains and plasmids used in the present study are listed in Table 1. Plasmids bearing proK and proL, the structural genes for tRNA1Pro and tRNA2Pro, respectively, were generously provided by Michael O'Connor (17).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Media and growth conditions. Cultures were grown in Vogel-Bonner minimal medium (22) supplemented with 0.2% glycerol and 0.05% acid casein hydrolysate (ACH), with or without 100 µg of L-tryptophan/ml. When necessary, one or more of the following antibiotics was added: ampicillin (50 µg/ml) or chloramphenicol (30 µg/ml). In some experiments 1% glucose was added to growing cultures. Bacterial growth was monitored by measuring the optical density at 600 nm (OD600).

RNA isolation and Northern blot analysis. To isolate total RNA from cells transformed with plasmid pGF25-14 or pGF25-00, cultures were grown in supplemented minimal medium, and cells were harvested in mid-log phase (OD600 = 0.8), collected by centrifugation, resuspended in a fresh ribosome isolation buffer (10 mM NH4Cl, 175 mM K acetate, 10 mM MgCl2, 35 mM Tris-Cl [pH 8.0], 1 mM dithiothreitol, 2 mM L-tryptophan), and disrupted by sonication. The resulting cell extracts were then centrifuged at 10,000 x g for 30 min to remove cell debris. Cleared extracts were extracted with acidic phenol (pH 4.2 to 5.1), the aqueous phase collected after centrifugation, and total RNA was isolated from the aqueous phase by ethanol precipitation. Where indicated, cell extracts were first treated with proteinase K (100 µg/ml, 37°C, 5 or 10 min) to release tRNAs from peptidyl-tRNAs before phenol extraction. Then, 20 µg of isolated RNA was electrophoresed on denaturing 6.5% polyacrylamide RNA gels. Separated RNA species were electroblotted onto Hybond-N+ membranes (Amersham Pharmacia). Northern blot hybridization was carried out by using 32P-end-labeled oligonucleotides specific to tRNA2Pro (5'-CCTCCGACCCCCGACACCCCAT-3') (5). Blots were visualized and quantified by using a PhosphorImager (Molecular Dynamics).

Detection of TnaC-tRNAPro in ribosomes in vivo. Cells were cultured in supplemented Vogel-Bonner minimal medium with appropriate antibiotics, with or without 100 µg of tryptophan/ml. Cells were harvested in mid-log phase (OD600 = 0.8), washed, and resuspended in fresh ribosome isolation buffer. All subsequent steps were performed at 4°C. Each cell suspension was sonicated, and the debris was removed by centrifugation for 30 min at 10,000 x g. The resulting extracts were either examined directly (total extracts) or centrifuged (for 2 h at 100,000 x g) to separate ribosome pellets from S-100 supernatants. Proteins and tRNAs in the various preparations were separated by electrophoresis on 10% Tricine-sodium dodecyl sulfate (SDS) protein gels, transferred to nylon membranes, and probed for TnaC-tRNA2Pro using the tRNA2Pro-specific oligonucleotide probes mentioned above. In detail, after cross-linking for 5 min with UV, membranes were prehybridized at 42°C for 1 h in a solution (10 ml per 10-by-5-cm membrane) consisting of 0.60 M NaCl, 0.12 M Tris-HCl, 8 mM Na2-EDTA (pH 8.0), 250 pg of sheared and denatured salmon sperm DNA/ml, 0.1% SDS, and 10x Denhardt solution (1x Denhardt solution = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone 40, and 0.02% Ficoll). Hybridization was at 42°C overnight in the same solution (10 ml) with the 5'32P-labeled oligonucleotide probes. Membranes were washed three times for 20 min each at 42°C with washing buffer (0.45 M NaCl, 0.09 M Tris-HCl, 6 mM Na2EDTA [pH 8.0], and 0.1% SDS) and then autoradiographed (21).


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RESULTS
 
Detection of TnaC-tRNA2Pro accumulation in vivo. Tryptophan codon 12 of tnaC is essential for tryptophan-induced accumulation of TnaC-tRNAPro in S-30 cell extracts (9). When tnaC is overexpressed from a multicopy plasmid (Fig. 1A), accumulation of TnaC-tRNA2Pro in vivo could conceivably lead to tRNA2Pro depletion, since this tRNA is solely responsible for translation of the proline codon that is the last codon of tnaC, CCU. To detect TnaC-tRNA2Pro accumulation in vivo, E. coli cells containing tnaC on a multicopy plasmid (Fig. 1A) were grown under inducing conditions, harvested by centrifugation, and disrupted by sonication, and Northern blotting was performed with 32P-labeled oligonucleotides specific for tRNA2Pro (see Materials and Methods). Our initial objective was to determine whether tRNA2Pro probing would detect tRNA2Pro as a component of TnaC-tRNA2Pro. The data in Fig. 1B demonstrate that TnaC-tRNA2Pro does accumulate, and is detectable, when wild-type tnaC is overexpressed in the presence of inducing levels of tryptophan (Fig. 1B, bottom panel, +Trp, lanes 7 and 9). In the absence of added tryptophan, only free tRNA2Pro was evident (Fig. 1B, upper panel, –Trp, all lanes). The TnaC-tRNA2Pro detected was shown to be exclusively associated with ribosomes; when ribosomes were separated from the supernatant by centrifugation, no TnaC-tRNA2Pro was detected in the S-100 supernatant; it was only in the pellet (bottom panel, compare lanes 8 and 9). As expected, TnaC-tRNA2Pro was not detected when the plasmid with W12R tnaC (the plasmid in which the Trp12 codon of TnaC was replaced by an Arg codon) was overexpressed in the presence of tryptophan (Fig. 1B, lanes 4, 5, and 6). Nor was TnaC-tRNA2Pro detected in preparations from cells with the vector-plasmid control (Fig. 1B, lanes 1, 2, and 3). Quantitation of the relative band intensities in Fig. 1B (lanes 7, 8, and 9) provided the estimate that when wild-type plasmid-borne tnaC was overexpressed in the presence of tryptophan, ~60% of the tRNA2Pro in the cells was present as TnaC-tRNA2Pro. Assuming there are ~720 copies of tRNA2Pro and ~5,000 ribosomes per cell (5), the number of tRNA2Pro molecules and fraction of ribosomes sequestered per cell would be ~430 and ~9.0%, respectively, under our experimental conditions.


Figure 1
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  		FIG. 1. (A) Schematic representation of plasmids pGF25-00 and pGF25-14. A 949-bp fragment containing the tna operon region from –213 to +306 from the tna promoter start site was joined to a fragment containing the rpoBC terminator, and this combined fragment was cloned into pUC18 (9). (B) The presence of inducing levels of tryptophan leads to TnaC-tRNA2Pro accumulation in vivo. E. coli cells bearing plasmid pUC18, pGF25-14 (W12R tnaC) or pGF25-00 (WT tnaC) were grown in a medium with or without 100 µg of L-tryptophan/ml (see Materials and Methods for details). To detect TnaC-tRNA2Pro in E. coli cultures, cells were harvested by centrifugation and disrupted by sonication, and total cellular extracts or ribosome pellets and S-100 supernatants were separated by centrifugation. Various fractions (50-µg samples of total extract protein and/or the corresponding fraction of supernatant and pellet) were analyzed for tRNA2Pro or peptidyl-tRNA2Pro after electrophoresis on a Tricine-SDS protein gel. Northern blotting was performed to detect tRNA2Pro using 32P-labeled oligonucleotides specific for tRNA2Pro. T, total extract; S, S-100 supernatant; P, ribosome pellet. The percentage of total tRNA2Pro present as TnaC-tRNA2Pro is shown.

Since electrophoresis on a Tricine-SDS protein gel was used to separate tRNA2Pro from TnaC-tRNA2Pro, the high pH of the buffer could have led to hydrolysis of some of the TnaC-tRNA2Pro that had accumulated. This would have reduced our estimate of the fraction of tRNA2Pro sequestered in ribosomes. To validate our quantitation, we used an additional procedure that would maintain the TnaC-tRNA2Pro stable during its isolation and separation. Phenol treatment of extracts is known to precipitate peptidyl-tRNAs, while tRNAs remain soluble. Upon acidic phenol extraction, the ester linkage in peptidyl-tRNAs remains stable. Strains overexpressing wild-type tnaC and W12R tnaC were grown in the presence of inducing levels of tryptophan, the cells were collected and were disrupted by sonication. Extracts containing ribosomes were then treated with phenol under acidic conditions to separate free tRNA2Pro from sequestered tRNA2Pro, i.e., TnaC-tRNA2Pro (Materials and Methods). After acidic phenol extraction, TnaC-tRNA2Pro was exclusively in the phenol phase (Fig. 2A, lane 2), whereas the majority of the free tRNA2Pro was soluble and was located in the aqueous phase (Fig. 2A, lane 3). To be certain that proteinase K could attack the TnaC-tRNA2Pro within intact ribosomes under our experimental conditions, cell extracts were treated with proteinase K before phenol extraction. Isolated material in the phenol phase and RNA in the aqueous phase were then analyzed after electrophoresis on 10% Tricine gels. Northern blot analyses indicated that the tRNA2Pro present initially as TnaC-tRNA2Pro was released by proteinase K and had moved to the aqueous phase (Fig. 2A, lanes 4 and 5). On the basis of these findings, some cell extracts were treated with proteinase K to release tRNA2Pro from TnaC-tRNA2Pro, and the resulting soluble cellular RNA was collected in the aqueous phase. Precipitated RNA (20 µg) from the aqueous phase was then electrophoresed on an acidic denaturing 6.5% polyacrylamide gel, electroblotted onto a Hybond-N+ membrane, and probed for tRNA2Pro (see Materials and Methods for details). 5S tRNA was also probed, and the 5S band was used as a reference in quantitating the relative levels of tRNA2Pro present. These results are shown in Fig. 2B. When wild-type tnaC was overexpressed, and extracts were not treated with proteinase K, only ca. 36% of the tRNA2Pro was present in the aqueous phase after phenol extraction (Fig. 2B, lane 4). Presumably, the remaining 64% of the tRNA2Pro was present in the phenol phase as TnaC-tRNA2Pro. Proteinase K treatment of cell extracts released the tRNA2Pro initially present as TnaC-tRNA2Pro (lanes 5 and 6). Under our experimental conditions, 5 min of digestion with proteinase K only partially released the tRNA2Pro present in TnaC-tRNA2Pro (lane 5); 10 min of proteinase K digestion appeared to release all of it (lane 6). In contrast, when the W12R mutant tnaC was overexpressed under the same conditions, all, or almost all, of the tRNA2Pro was found in the aqueous phase (Fig. 2B, lane 1). Proteinase K treatment of the cell extracts with W12R tnaC did not release any tRNA2Pro; thus, there apparently was no TnaC(W12R)-tRNA2Pro (Fig. 2B, lanes 2 and 3). These results suggest that the fraction (>60%) of tRNA2Pro retained in the phenol phase after expression of wild-type tnaC is covalently attached to TnaC, i.e., as TnaC- tRNA2Pro (Fig. 2B, lane 4). It is unlikely that there was any other peptidyl-tRNA with tRNA2Pro, since when W12R tnaC was overexpressed almost all of the tRNA2Pro was present in the aqueous phase after phenol extraction (Fig. 2B, lanes 1 to 3). These data agree with our calculations in Fig. 1 and confirm that when wild-type tnaC is overexpressed in the presence of tryptophan, more than 60% of the tRNA2Pro in the cell is sequestered as TnaC-tRNA2Pro.


Figure 2
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  		FIG. 2. Quantitation of the relative level of tRNA2Pro sequestered as TnaC-tRNA2Pro during tnaC overexpression in vivo. (A) E. coli cells bearing pGF25-14 (W12R tnaC) or pGF25-00 (wild-type [WT] tnaC) were grown in a medium with 100 µg of L-tryptophan/ml. Cells were harvested, disrupted by sonication, and centrifuged to remove debris, and extracts were either mock treated or treated with proteinase K at 37°C for 5 min or 10 min. Total protein-containing fractions and RNA from cells harboring pGF25-00 (WT tnaC) were isolated by using acidic phenol treatment, and components were analyzed by electrophoresis on 10% Tricine gels. Northern blotting was performed by using anti-tRNA2Pro probes. (B) The components in 20 µg of RNA from each sample were separated by electrophoresis on denaturing 6.5% polyacrylamide RNA gels, and Northern blotting hybridization analyses were performed with 5' 32P-end-labeled oligonucleotides specific for tRNA2Pro or tRNA2Pro-containing compounds. 5S tRNA was probed by using an appropriate labeled oligonucleotide and was used as a loading control. The ratios of counts in the tRNA2Pro band to counts in the 5S tRNA band are shown. Relative levels of tRNA2Pro were normalized to the tRNA2Pro from cells harboring pGF25-14 (W12R tnaC) without treatment with PK, which was set at 1.0.

Estimation of the in vivo stability of overproduced TnaC-tRNA2Pro. There are various mechanisms that the cell might use to cleave accumulated TnaC-tRNA2Pro and release the stalled ribosomes and provide free tRNA2Pro for general protein synthesis. Experiments were therefore performed to examine the in vivo stability of accumulated TnaC-tRNA2Pro. Using our plasmid-based tnaC overexpression system, TnaC-tRNA2Pro was overproduced in a growing culture, and then glucose was added to activate catabolite repression and shut off tna operon transcription. Glucose addition to cultures containing glycerol and tryptophan is known to reduce tna operon expression by >100-fold (19). Samples were removed at various times, and the stability of the previously synthesized TnaC-tRNA2Pro was determined (Fig. 3). It can be seen that the accumulated TnaC-tRNA2Pro was somewhat unstable; it had a half-life of 10 to 15 min under the conditions examined. These findings demonstrate that this peptidyl-tRNA is sufficiently stable in vivo that its synthesis and survival could deplete the pool of free tRNA2Pro needed for additional protein synthesis.


Figure 3
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  		FIG. 3. Stability in vivo of overproduced TnaC-tRNA2Pro. (A) Detection of TnaC-tRNA2Pro after glucose addition to a culture in vivo. Cells bearing plasmid pGF25-00 were grown in minimal medium supplemented with 0.05% ACH and 0.2% glycerol with 100 µg of L-tryptophan/ml. Cell growth was monitored by measuring OD600. A total of 1.0% glucose was added at an OD600 of 0.6. Samples were taken at the indicated times (in minutes). After sonication and centrifugation, samples containing 50 µg of protein were analyzed for tRNA2Pro or peptidyl-tRNA2Pro after electrophoresis on a Tricine-SDS gel. Northern blotting was performed to detect tRNA2Pro using 32P-labeled oligonucleotides specific for tRNA2Pro. The percentage of total tRNA2Pro present as TnaC-tRNA2Pro is shown. (B) Time course of TnaC-tRNA2Pro decay calculated from the data in panel A.

Growth rate inhibition by tnaC overexpression and its relief by increased tRNAPro production. If the cellular level of free tRNA2Pro is appreciably depleted by tnaC overexpression, this might reduce the cell growth rate. To explore this possibility, the cell growth rates of relevant strains were determined in media with or without added tryptophan. The cell growth rate was severely reduced when wild-type W12 tnaC was overexpressed in the presence of inducing levels of tryptophan (Fig. 4A, compare pGF25-00 –Trp with pGF25-00 +Trp). When the W12R tnaC construct or the empty vector was introduced into E. coli (Fig. 4A, pUC and p25-14) the cultures grew normally, with or without added tryptophan. No growth rate reduction was observed with the E. coli wild-type strain W3110 or the mutant lacking tryptophanase, W3110 tnaA2, with or without added tryptophan (Fig. 4A).


Figure 4
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  		FIG. 4. Growth inhibition and tRNA2Pro overproduction in E. coli. (A) Cells bearing the different plasmids shown in Fig. 4A were grown in minimal medium supplemented with 0.05% ACH and 0.2% glycerol with or without 100 µg of L-tryptophan/ml. Cell growth was monitored by measuring OD600. (B) Measurement of tRNA2Pro levels in cultures containing different plasmids. E. coli cells containing pProL5 (tRNA2Pro) or pHSG575 (vector) were grown in minimal medium supplemented with 0.05% ACH and 0.2% glycerol, without 100 µg of tryptophan/ml, in the presence of chloramphenicol (30 µg/ml). Supernatants and ribosomes were separated by centrifugation, samples were electrophoresed, and the components were probed using tRNA2Pro- and 5S tRNA-specific oligonucleotide probes. (C) E. coli cells bearing plasmids pGF25-00 (WT tnaC) and pRroL5 (tRNA2Pro) or pHSG575 (vector) were grown in medium with 100 µg of tryptophan/ml. S-100 supernatants and pellets were treated as described in Materials and Methods to measure the presence of TnaC-tRNA2Pro and/or tRNA2Pro in the supernatant or ribosome pellet. The percentage of total tRNA2Pro present as TnaC-tRNA2Pro is shown.

On the basis of our estimate of the extent of tRNA2Pro sequestration in TnaC-tRNA2Pro when wild-type tnaC is overexpressed from a multicopy plasmid, we reasoned that depletion of free tRNA2Pro is the likely cause of growth rate reduction. We therefore tested whether overexpression of tRNA2Pro could relieve the growth inhibition associated with tnaC overexpression. Initially, we examined the extent of plasmid-based tRNA2Pro overexpression in E. coli W3110 tnaA2. Figure 4B shows that the tRNA2Pro level in cells containing the tRNA2Pro overexpression plasmid, pProL5, was at least five times higher than in cells with pHSG575, the control, empty plasmid. As expected, additional tRNA2Pro was found in both the supernatant and the ribosome pellet (Fig. 4B and 4C). We then examined TnaC-tRNA2Pro accumulation in tryptophan-induced E. coli cultures overproducing both tRNA2Pro and tnaC. We observed that in the presence of elevated levels of tRNA2Pro, the extent of accumulation of TnaC-tRNA2Pro was dependent on the presence of inducing levels of tryptophan; ~2-fold more TnaC-tRNA2Pro was observed per cell when tRNA2Pro was overexpressed (Fig. 4C, lane P with pProL5 versus lane P with vector). It was obvious that even when TnaC-tRNA2Pro was accumulated in the presence of pProL5, a large amount of free tRNA2Pro was present (Fig. 4C, lane S, pProL5 panel). When tRNA2Pro was overproduced, the percentage of tRNA2Pro as TnaC-tRNA2Pro decreased to 12.3% (Fig. 4C, left panel). In contrast, more than 63% of tRNA2Pro was associated with TnaC-tRNA2Pro when tRNA2Pro was not overproduced, and the parental plasmid was present (Fig. 4C, right P panel).

Having confirmed that tRNA2Pro is overexpressed in strains with pProL5, we next sought to determine whether this overexpression would relieve the observed growth inhibition. In Fig. 5A it is shown that overexpression of tRNA2Pro did reverse the growth inhibition attributed to wild-type tnaC overexpression (Fig. 5A). Neither overexpression of tRNA1Pro nor the presence of the tRNA vector plasmid overcame this growth inhibition (Fig. 5A). Growth inhibition by tnaC overexpression was also evident when the Pro24 codon of tnaC, CCU, was replaced by the Pro24 codon, CCG, a codon specifically translated by tRNA1Pro (Fig. 5B). It has been estimated that there are ~900 molecules of tRNA1Pro per E. coli cell (5). As expected, TnaC-tRNA1Pro also accumulated when tnaC (CCG) was overexpressed in the presence of inducing levels of tryptophan (data not shown). Significantly, overexpression of tRNA1Pro, rather than tRNA2Pro, relieved this growth inhibition (Fig. 5B). These data demonstrate that sequestration of the specific tRNAPro that decodes the last sense codon of tnaC, as TnaC-tRNAPro, in the stalled ribosome complex, causes the growth inhibition associated with tnaC overexpression.


Figure 5
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  		FIG. 5. Overexpression of the tRNAPro that can translate the last sense codon of tnaC relieves growth inhibition caused by tnaC overexpression in the presence of tryptophan. E. coli cells bearing the different plasmids listed in the figure (also see Table 1) were grown in minimal medium supplemented with 0.05% ACH and 0.2% glycerol, with or without 100 µg of L-tryptophan/ml, and with shaking at 37°C. Cell growth was monitored at OD600. (A) Cells with the plasmid overexpressing wild-type tnaC plus the parental plasmid, the plasmid encoding tRNA2Pro (pProL5), or the plasmid expressing tRNA1Pro (pProK4), were grown in the absence or presence of tryptophan. (B) Cells with the plasmid overexpressing tnaC with the Pro24 codon CCG, plus the parental plasmid, the plasmid encoding tRNA1Pro (pProK4), or the plasmid encoding tRNA2Pro (pProL5), were grown in the absence or presence of tryptophan.


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DISCUSSION
 
Using an E. coli S-30 in vitro system it has been shown that tryptophan induction of tna operon expression leads to inhibition of RF2-mediated cleavage of TnaC-tRNA2Pro (8). The translating ribosome appears to stall at the tnaC stop codon, and this stalling blocks Rho factor's access to its rut binding site located on the tna mRNA in the vicinity of the tnaC stop codon. This prevents Rho-dependent transcription termination in the leader region of the tna operon (8). To validate this model, it was critical to show in vivo that inducing levels of tryptophan in the growth medium do result in TnaC-tRNAPro accumulation, and ribosome stalling. Previous attempts to detect TnaC-tRNAPro in vivo were unsuccessful (data not shown), leaving open the possibility that some mechanism of peptidyl-tRNA cleavage is operable within the growing cell.

In the present study, we developed a novel method to monitor TnaC-tRNA2Pro accumulation in vivo. Cultures containing a multicopy plasmid bearing tnaC were grown under inducing and noninducing conditions, and cell extracts were prepared. Ribosome pellets were separated from supernatants, tRNA2Pro and TnaC-tRNA2Pro were separated by gel electrophoresis, and the separated material was transferred to a nylon membrane. Northern blotting was performed to detect tRNA2Pro, both as free tRNA2Pro and as TnaC-tRNA2Pro, by using 32P-labeled oligonucleotides specific to tRNA2Pro (see Materials and Methods for details). Using this procedure, tryptophan-induced accumulation of TnaC-tRNA2Pro was observed in vivo (Fig. 1 and 4C). Moreover, ca. 60% of the tRNA2Pro in the cells was present as TnaC-tRNA2Pro. The TnaC-tRNA2Pro detected was exclusively associated with ribosome pellets (Fig. 1 and 4C). In addition, the total amount of tRNA2Pro in cells appeared to be unchanged even when more than ca. 60% of the tRNA2Pro was sequestered as TnaC-tRNA2Pro (Fig. 2B, lanes 1 and 6). Apparently, E. coli lacks a mechanism for increasing tRNA2Pro synthesis or release when free tRNA2Pro is depleted by tnaC overexpression.

When tnaC was overexpressed in the presence of tryptophan, both the growth rate and the rate of overall protein synthesis were reduced. Since tryptophan induces TnaC-tRNA2Pro accumulation in vivo (Fig. 1), tRNA2Pro sequestration was considered the likely cause of this inhibition. Overexpression of tRNA2Pro did in fact largely relieve this growth inhibition. Furthermore, when the Pro24 codon of tnaC, CCU, was replaced by CCG, and tnaC Pro24 CCG was overexpressed in the presence of inducing levels of tryptophan, overexpression of tRNA1Pro, rather than tRNA2Pro, relieved this tnaC Pro24 CCG inhibition (Fig. 5). In these experiments, TnaC-tRNA1Pro accumulation was also observed (data not shown). These data establish that depletion of the tRNA reading the last sense codon of tnaC is primarily responsible for the inhibition of cell growth associated with tryptophan induction.

Multicopy plasmids carrying tnaC in a strain with a chromosomal tnaA'-'lacZ fusion have been shown to inhibit tryptophan-induced TnaA-LacZ (ß-galactosidase) production (6). Mutational studies established that this inhibition was not due to inhibition of transcription initiation, translation initiation, tryptophan transport, or enzyme activity (6). On the basis of our findings, we believe that this inhibition was a result of tRNA2Pro depletion. In fact, we have found that overproduction of tRNA2Pro does restore TnaA-LacZ production by the chromosomal operon (data not shown).

Inhibition of cell growth by tnaC overexpression mimics the inhibitory effect conferred by minigene expression (14, 20). However, the exact mechanisms causing tRNA depletion differ in these two systems. The deleterious effect of minigene expression is mediated by depletion of the corresponding pools of free tRNAs (14, 20). Inhibition occurs because cells fail to recycle efficiently the tRNAs from the peptidyl-tRNAs released from ribosomes, causing starvation for essential species of tRNAs. Thus, peptidyl-tRNA dropoff and inefficiency of tRNA recycling catalyzed by Pth are essential features of this minigene-conferred inhibition. Regarding growth inhibition by tnaC overexpression, our estimate of the stability of TnaC-tRNA2Pro within the cells of an induced culture suggests that it has a half-life of about 10 to 15 min (Fig. 3). It appears that TnaC-tRNA2Pro is retained within translating ribosomes for a period sufficiently long to create a free tRNA2Pro deficiency.


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ACKNOWLEDGMENTS
 
We are grateful to Michael O'Connor for providing plasmids that overexpress genes encoding tRNAPro. We also thank Roger Cruz-Vera for advice during the course of this study and Valley Stewart for very helpful comments on the manuscript.

These studies were supported by a grant (to C.Y.) from the National Science Foundation (MCB-0093023).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Phone: (650) 725-1835. Fax: (650) 725-8221. E-mail: yanofsky{at}cmgm.stanford.edu. Back

{dagger} Present address: Department of Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660. Back


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REFERENCES
 
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Journal of Bacteriology, March 2006, p. 1892-1898, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1892-1898.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Stewart, V. (2008). The Ribosome: a Metabolite-Responsive Transcription Regulator. J. Bacteriol. 190: 4787-4790 [Full Text]  
  • Yanofsky, C. (2007). RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria. RNA 13: 1141-1154 [Abstract] [Full Text]  
  • Gong, M., Cruz-Vera, L. R., Yanofsky, C. (2007). Ribosome Recycling Factor and Release Factor 3 Action Promotes TnaC-Peptidyl-tRNA Dropoff and Relieves Ribosome Stalling during Tryptophan Induction of tna Operon Expression in Escherichia coli. J. Bacteriol. 189: 3147-3155 [Abstract] [Full Text]  
  • Cruz-Vera, L. R., Gong, M., Yanofsky, C. (2006). Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide. Proc. Natl. Acad. Sci. USA 103: 3598-3603 [Abstract] [Full Text]  

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