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Contributed by Charles Yanofsky, January 13, 2006
Abstract
Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNAPro, the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNAPro peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNAPro complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNAPro or its nonfunctional substitute, TnaC(W12R)-tRNAPro. Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNAPro complexes but not of ribosome-TnaC(W12R)-tRNAPro complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNAPro; it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNAPro mutant complexes. These findings establish that Trp-12 of TnaC-tRNAPro is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.
The Escherichia coli tna operon consists of a promoter-leader regulatory region followed by the structural genes tnaA and tnaB, encoding, respectively, the enzyme tryptophanase and a tryptophan-specific permease (1). Tryptophanase catalyzes the reversible hydrolysis of tryptophan to indole, pyruvate, and ammonia. Transcription of the structural genes of the tna operon is regulated by both catabolite-repression and tryptophan-induced inhibition of Rho factor-dependent transcription termination in the leader region of the operon (2, 3). The transcript of the tna operon leader region contains a coding region for a 24-residue leader peptide, tnaC, followed by a Rho factor-binding site. In the presence of free tryptophan, cleavage of the nascent peptidyl tRNA, TnaC-tRNAPro, at its UGA stop codon, is inhibited (4). This peptidyl-tRNA therefore remains uncleaved, within the translating ribosome, resulting in ribosome stalling at the tnaC stop codon on the tna transcript. The stalled ribosome blocks Rho factor’s access to its rut RNA-binding site located immediately following the tnaC stop codon, thereby preventing Rho-catalyzed transcription termination in the leader region of the operon (5). Several of the features of TnaC-tRNAPro required for inhibition of its cleavage have been identified: the crucial tryptophan residue at position 12, the last amino acid in the peptide, Pro-24, and the spacing between Trp-12 and Pro-24 (3, 6). Features of the ribosome required for induction have also been identified: Lys-90 of ribosomal protein L22 and nucleotide U2609 of 23S rRNA are essential. Both are located in the ribosome exit tunnel, near the putative position of Trp-12 of TnaC-tRNAPro (7).
Other nascent peptides are known to act in cis, much like TnaC, to inhibit completion of synthesis of a nascent peptide (8–13). Examples are secM of E. coli (8) and cytomegalovirus UL4 (14). Neither of these requires an additional factor to inhibit the peptidyl transferase reaction and induce ribosome stalling. There are other examples like the tna operon, where the presence of a small molecule also is required for ribosome stalling. The antibiotic chloramphenicol induces ribosome stalling during translation of the leader peptide coding region preceding the chloramphenicol resistance gene (9). Spermine or spermidine induces stalling in a small ORF regulating synthesis of S-adenosylmethionine decarboxylase (12). In addition, high concentrations of arginine are required for ribosome stalling during synthesis of a fungal leader peptide, AAP (13). In these examples it has been suggested that the nascent peptidyl-tRNA, either alone or with the aid of an accessory molecule, promotes changes in the polypeptide exit tunnel that influence amino acid addition or peptidyl-tRNA cleavage at the peptidyl transferase center.
Studies in vitro with the tna operon have established that free tryptophan binds to the ribosome synthesizing TnaC-tRNAPro, inhibiting Release Factor 2 (RF2)-promoted cleavage of this peptidyl-tRNA at the tnaC UGA stop codon (4). The precise site of binding of free tryptophan in the TnaC-tRNAPro-ribosome complex has not been reported. However, it has been shown that when the tnaC UGA stop codon is replaced by a tryptophan codon, tryptophan-charged RNA Trp can substitute for tryptophan as inducer and inhibit TnaC-tRNAPro cleavage or elongation (15). This finding suggested that the region of the ribosomal A site occupied by the tryptophanyl moiety of tryptophanyl-tRNATrp may be the site of free tryptophan binding and action (15).
In the current study, in vivo and in vitro experimental approaches were used to determine the likely location of the free tryptophan binding site and the role of the nascent TnaC-tRNAPro in free tryptophan binding and action. Ribosome-TnaC-tRNAPro complexes were produced in vitro and isolated by using biotin-labeled tna operon leader transcripts, encoding TnaC with its normal key residue, Trp-12, or TnaC with its nonfunctional Trp-12 to Arg-12 substitute. The isolated complexes were then examined in methylation protection and competition assays, with and without tryptophan, noninducing tryptophan analogs, or antibiotic inhibitors of the peptidyl transferase reaction. Changes in the ribosome were identified; the inducer, free tryptophan, caused these changes when it was bound to ribosomes containing TnaC-tRNAPro.
Results
Addition of Free Tryptophan Inhibits Transfer of the TnaC Peptide of TnaC-tRNAPro, but Not of TnaC (W12R)-tRNAPro, to Puromycin or Aminoacyl-tRNA.
Tryptophan has been shown to inhibit the cleavage of TnaC-tRNAPro by puromycin (4). Competition assays using isolated wild-type ribosome-TnaC-tRNAPro complexes treated with anti-RF2 antibodies revealed that free tryptophan is effective as a competitive inhibitor of puromycin action (Fig. 1 A and B). In the absence of added tryptophan, addition of puromycin at concentrations between 0.02 and 2 mM resulted in >90% hydrolysis of TnaC-tRNAPro, in a solution with 50 nM stalled ribosomes (Fig. 1 B, filled squares). However, when 2 mM tryptophan was also present, puromycin was much less effective in cleaving TnaC-tRNAPro; a concentration of 2 mM was required to reverse the effects of the 2 mM tryptophan (Fig. 1 B, open squares). Comparable analyses performed with ribosome-TnaC(W12R)-tRNAPro mutant complexes revealed that tryptophan was incapable of preventing puromycin action (Fig. 1 A and B). Competition analyses were also performed by using different concentrations of tryptophan and a constant, low concentration of puromycin (0.02 mM). This level of puromycin did not induce hydrolysis when present with 2 mM tryptophan (Fig. 1 A and B, open squares). Tryptophan concentrations of 0.1 mM and below did not prevent puromycin action (Fig. 1 C and D, open circles). Most importantly, tryptophan, even at the highest concentration tested, was incapable of inhibiting puromycin hydrolysis of the mutant peptidyl-tRNA, TnaC(W12R)-tRNAPro (Fig. 1 C and D, filled circles). These data establish that the W12R change in TnaC appears to prevent tryptophan from binding to the ribosome or influencing puromycin binding and action.
Fig. 1.
Analysis of tryptophan inhibition of puromycin cleavage of ribosome complexes containing wild-type TnaC-tRNAPro or mutant TnaC(W12R)-tRNAPro. Complexes formed with either wild-type tnaC mRNA or mutant tnaC (W12R) mRNA, prepared in cell-free extracts pretreated with anti-RF2 antibodies, were isolated by using streptavidin beads. These bead-bound complexes were washed and resuspended in the presence (+) or absence (−) of 2 mM tryptophan (Trp). (A) Mixtures with or without tryptophan were incubated with different concentrations of puromycin (Puro) for 7 min at 37°C. Reaction products were then resolved by electrophoresis in 10% Tris-Tricine polyacrylamide gels and transferred to nitrocellulose membranes. Northern blot assays were used to detect and measure the levels of wild-type and mutant peptidyl-tRNA by using a 32P-labeled oligonucleotide complementary to tRNAPro. (B) Curves displaying the extent of hydrolysis of the two peptidyl-tRNAs examined in A. (C) Isolated bead complexes were washed with a solution lacking tryptophan and were then incubated with increasing concentrations of tryptophan for 5 min at 37°C. Puromycin was (+) or was not (−) added, and the peptidyl-tRNA level was determined as described in A. (D) Curves showing the extent of hydrolysis of the peptidyl-tRNAs displayed in C. The percent value shown represents the cpm in each peptidyl-tRNA in the “+” tryptophan lane divided by the cpm in the peptidyl-tRNA in the lane minus puromycin. Each experiment was performed three times.
The effectiveness of tryptophan as an inhibitor of peptidyl-tRNA hydrolysis could depend on the molecule mediating hydrolysis, e.g., a release factor, puromycin, or an aminoacyl-tRNA. To determine whether tryptophan also inhibits aminoacyl-tRNA interaction, several relevant changes were introduced into the tnaC coding region. An isoleucine codon, AUA or AUU, was inserted between the last sense codon of tnaC, proline CCU, and the tnaC stop codon, UGA. AUA is a rarely used isoleucine codon translated by a scarce tRNA, present at <100 molecules per cell (16). AUU is a common isoleucine codon, decoded by a tRNA present at 4,000 molecules per cell (16). After overexpression of these mutant tnaC genes in vivo, in the presence or absence of tryptophan, the extent of accumulation of TnaC-tRNA2 Pro was determined by Northern blot analysis (see Materials and Methods). Using the tnaC-AUA construct, we observed that addition of tryptophan led to an appreciable accumulation of TnaC-tRNAPro (compare lanes 1 and 2 in Fig. 2). When the tnaC-AUU construct was examined, tryptophan addition had essentially no effect; there was no accumulation of TnaC-tRNAPro (compare lanes 3 and 4 in Fig. 2). Interestingly, no TnaC (W12R)-tRNAPro was accumulated when the AUA codon was added following proline codon 24 of the transcript specifying this peptidyl-tRNA (compare lanes 5 and 6 in Fig. 2). This result indicates that tryptophan action with a tna transcript bearing this rare codon depends on the presence of Trp-12 of TnaC-tRNAPro. Apparently the existence of a codon decoded by a scarce tRNA following proline codon 24 of tnaC allows tryptophan to bind to the ribosome and inhibit its peptidyl transferase activity. A binding site for tryptophan therefore appears to be created in the ribosomal A site by the features of TnaC-tRNAPro, and the bound tryptophan can inhibit the action of RF2, or puromycin, at the UGA stop codon, or inhibit the reaction with an aminoacyl-tRNA at a rare sense codon.
Fig. 2.
Analysis of TnaC-tRNAPro and TnaC (W12R)-tRNAPro accumulation after tryptophan induction in vivo of wild-type or mutant tnaC genes with the added isoleucine codon, AUA25 or AUU25. Cultures of strain W3110 (tnaA2) transformed with plasmid pCV25–00A25 (tnaC-AUA25), pCV25–00U25 (tnaC-AUU25), or pCV25–14A25 [tnaC (W12R)-AUA25] were grown in the presence (+) or absence (−) of 100 μg/ml tryptophan. Clarified cell extracts were prepared and subjected to electrophoresis as described in Materials and Methods, and the peptidyl-tRNA TnaC-tRNAPro and tRNAPro levels were detected as described in Fig. 1 A. Each experiment was performed three times.
Free Tryptophan Binding Affects Methylation of A Site Nucleotide A2572 of Isolated Complexes with TnaC-tRNAPro but Not with TnaC(W12R)-tRNAPro.
The findings in the previous section suggest that free tryptophan can bind at the ribosomal A site and inhibit peptidyl transferase activity. Next we wanted to determine whether free tryptophan affects the physical positioning or pairing of 23S rRNA nucleotides that are believed to constitute the peptidyl transferase center (17). We had shown previously that isolated ribosome-TnaC-tRNAPro complexes could be used to detect changes in the methylation pattern of 23S rRNA nucleotides in these complexes (7). In the present study methylation protection assays were performed with TnaC-tRNAPro and TnaC(W12R)-tRNAPro complexes treated with anti-RF2 antibodies (Fig. 3 B). Solutions containing isolated bead complexes prepared with tnaC mRNAs were treated with the methylation agent dimethyl sulfate (DMS). A significant reduction was observed in the methylation of nucleotide A2572 when tryptophan was present with the TnaC-tRNAPro complex (compare lanes 2 and 3 in Fig. 3 B). We did not observe a change in the methylation intensity of nucleotide A2572 when tryptophan was added to the TnaC(W12R)-tRNAPro complex (compare lanes 4 and 5 in Fig. 3 B). Additionally, no reduction in methylation of A2572 was observed when tryptophan was added to free ribosomes (compare lanes 2 and 3 in Fig. 3 A). The methylation intensity of nucleotide A2602 was not affected by the presence of tryptophan and was identical under all conditions (Fig. 3 A and B).
Fig. 3.
Analyses of methylation changes in 23S rRNA induced by free tryptophan, with TnaC-tRNAPro or TnaC(W12R)-tRNAPro complexes treated with anti-RF2 antibodies. (A) Ribosomes isolated from cell-free extracts were incubated in the presence (+) or absence (−) of tryptophan (Trp) for 5 min at 37°C. The solutions were mixed with 1/150 (vol/vol) of the methylation agent DMS and incubated at room temperature for 10 min. Total RNA was extracted and used in primer extension assays with a 32P-labeled oligonucleotide complementary to nucleotides 2654–2674 of 23S rRNA. The extension assays were resolved by electrophoresis in 7 M urea/6.5% polyacrylamide gels. The nucleotides indicated on the left were methylated by DMS. (B) Complexes bound to streptavidin beads were isolated as described in Fig. 1. The beads were washed and resuspended in a solution without tryptophan. These mixtures were then incubated with (+) or without (−) tryptophan for 5 min at 37°C. The 23S rRNA in each sample was methylated with DMS, and the rRNA nucleotides resolved as indicated above. Primer extension analyses were performed by using the various 23S rRNAs and a 32P-labeled oligonucleotide complementary to 23S rRNA nucleotides 2654–2674. For nucleotide A2572, the percent methylation shown corresponds to the ratio of the cpm detected for the experimental A2572 band divided by the cpm for the band obtained with the control sample of Ribo-tnaC mRNA without tryptophan. (C) Samples of an isolated bead complex in a solution without tryptophan were incubated with (+) or without (−) tryptophanol (Trp-OH) for 5 min at 37°C. Tryptophan was then added, and the mixtures were incubated an additional 10 min. The mixtures were finally incubated in the presence (+) or absence (−) of puromycin for 5 min. The reaction products were separated by electrophoresis and the peptidyl-tRNA TnaC-tRNAPro was measured as described in Fig. 1. (D) The various bead complexes were washed and resuspended in a solution without tryptophan. They were then incubated with (+) or without (−) tryptophanol (Trp-OH) for 5 min at 37°C, after prior incubation for 5 min in the presence (+) or absence (−) of tryptophan (Trp). The 23S rRNA was methylated with DMS, and the labeled bands were resolved and identified. The percent methylation was calculated as indicated above. Each experiment was performed four times. The band corresponding to nucleotide A2602 was used as a methylation control and for standardizing recovery.
Protection of nucleotide A2572 from methylation by the presence of tryptophan could be due to the interaction of tryptophan with its ribosomal binding site or the changes it introduces in the peptidyl transferase center. To address these possibilities we examined the effects of the presence of tryptophanol, a non-inducer and competitor of tryptophan. As shown in Fig. 3 C, tryptophanol did not inhibit the hydrolysis of TnaC-tRNAPro by puromycin (compare lanes 1 and 3). However, tryptophanol addition did reverse the ability of tryptophan to protect TnaC-tRNAPro from puromycin hydrolysis (compare lanes 4 and 5 in Fig. 3 C). This finding implies that tryptophanol can effectively compete with tryptophan for ribosome binding; competition was demonstrated previously with another tryptophan analog, tryptamine (4). Methylation-protection assays with isolated complexes were also performed in the presence of tryptophanol. As shown in Fig. 3 D, tryptophanol, unlike tryptophan, did not reduce the methylation of nucleotide A2572 of wild-type complexes (compare lanes 2–4 in Fig. 3 D). However, tryptophanol added with tryptophan did prevent tryptophan from inhibiting the methylation of A2572 (compare lanes 3 and 5 in Fig. 3 D). This result implies that tryptophanol can occupy the tryptophan-binding site. When it does bind, it does not protect nucleotide A2572 from methylation, nor does it produce the changes in the ribosome required for operon induction.
Free Tryptophan Bound to TnaC-tRNAPro-Ribosome Complexes Affects Antibiotic Interaction at the Peptidyl Transferase Center.
Several antibiotics are known to inhibit puromycin action or aminoacyl-tRNA reactivity within the ribosome (18). Competition assays were therefore performed to determine whether tryptophan would inhibit antibiotic interaction with the ribosome. TnaC-tRNAPro complexes that had been pretreated with anti-RF2 antibodies were examined, using a methylation-protection assay. The antibiotics chloramphenicol and sparsomycin were used, because these had been shown to inhibit puromycin-induced hydrolysis of TnaC-tRNAPro in our system (data not shown). The addition of these antibiotics to isolated complexes enhanced the methylation of nucleotide A2058 or A2059 (Fig. 4 A: for chloramphenicol, compare lane 2 with lanes 4 and 10; for sparsomycin, compare lane 2 with lanes 6 and 12). The presence of tryptophan alone did not affect methylation of these nucleotides (compare lanes 2 and 3 in Fig. 4 A). The enhancement of methylation of nucleotide A2058 induced by chloramphenicol addition was not reduced by the presence of tryptophan, in either the wild-type or mutant complexes examined (compare lane 4 with lane 5, and lane 10 with lane 11 in Fig. 4 A). These results suggest that tryptophan does not affect chloramphenicol interaction with the ribosome. The minor increase in the methylation of nucleotide A2058 that occurred in the presence of sparsomycin was not affected by tryptophan, with either TnaC-tRNAPro or TnaC (W12R)-tRNAPro ribosome complexes (Fig. 4 A and B). However, the increase in methylation of nucleotide A2059 produced by sparsomycin’s presence was reduced by the addition of tryptophan to the isolated TnaC-tRNAPro complexes [compare lane 6 with lane 7 in Fig. 4 A (numbers underlined)]. This protection by tryptophan was not observed with mutant TnaC(W12R)-tRNAPro complexes (compare lane 12 with lane 13 in Fig. 4 A). Thus, tryptophan does affect the ability of sparsomycin to interact with the peptidyl transferase center. We also found that tryptophanol, unlike tryptophan, does not appreciably affect the sparsomycin enhancement of methylation of nucleotide A2059 of TnaC-tRNAPro (compare lane 4 with lane 5 in Fig. 4 B). Thus, tryptophanol can effectively compete with tryptophan for ribosome binding (Fig. 3 C), but unlike tryptophan, it does not appear to compete with sparsomycin (Fig. 4 B). These results suggest that the tryptophan-binding site is not identical to the sparsomycin-binding site. However, these sites may be close or may overlap.
Fig. 4.
Analysis of methylation changes in 23S rRNA produced by the addition of antibiotics to isolated ribosome complexes. Complexes were isolated as described in the legend to Fig. 1. (A) Complexes containing wild-type tnaC mRNA or tnaC(W12R) mRNA bound to streptavidin beads were isolated, washed, resuspended, and incubated with (+) or without (−) tryptophan for 5 min at 37°C. Inhibitors of translation were then added at the concentrations indicated, and the mixtures were incubated for an additional 10 min. The 23S rRNA was then methylated by using DMS, and the methylated nucleotides were detected as described in Fig. 3. Primer extension assays were performed by using a 32P-labeled oligonucleotide complementary to nucleotides 2102–2122 of 23S rRNA. (B) Isolated complexes with wild-type tnaC mRNA that had been washed to remove free tryptophan were incubated with (+) or without (−) tryptophanol (Trp-OH) for 5 min at 37°C, after which they were incubated with (+) or without (−) sparsomycin for an additional 10 min. The samples were then treated with DMS, and nucleotide methylation was analyzed as described above. The percent methylation of nucleotides A2058 and A2059 was calculated as described in Fig. 3. Each experiment was performed three times.
Discussion
The objectives of our current studies are to determine the features of the ribosome, ribosome-bound TnaC-tRNAPro, and free tryptophan that are responsible for tryptophan induction of tna operon expression. Recent structural analyses with the E. coli 70S ribosome have described the locations of the residues that form the peptide exit tunnel, the A and P sites, and the peptidyl transferase center (19). Our studies have examined some of these residues for their possible role(s) in recognition of the features of nascent TnaC-tRNAPro and free tryptophan that are responsible for inhibition of peptidyl transferase activity. Cross-linking analyses with Lys-11 of TnaC-tRNAPro revealed that this residue cross-links to nucleotide A750 (Fig. 5, pink residue) (7). It was also observed that Trp-12 of ribosome-bound TnaC-tRNAPro protects nucleotide A788 from methylation (Fig. 5, orange residue) (7). This residue is in the peptide exit tunnel. In vivo studies (7) revealed that substitutions of residue K90 of the L22 protein, a residue within the segment of this polypeptide that enters the exit tunnel, reduces or eliminates tna operon induction (Fig. 5, green residue). It was also found that several nucleotide changes in 23S rRNA reduce or prevent induction (7): U2609C (Fig. 5, brown residue), U754A (Fig. 5, gray residue), and the addition of an A residue at position 751 (Fig. 5, site of likely insertion identified). These findings establish the likely location of Trp-12 of TnaC-tRNAPro in the ribosome exit tunnel and identify nearby residues that influence induction (Fig. 5, see Trp-12) (7).
Fig. 5.
Regions of the E. coli 50S ribosome subunit believed to play a role in tryptophan induction of tna operon expression. This figure is based on the structure determined by Schuwirt et al. (19). Identified are nucleotides of 23S rRNA and residue K90 of the L22 ribosomal protein that are required for tryptophan inhibition of TnaC-tRNAPro hydrolysis. Also indicated are the putative locations of (i) the tRNAPro (curved purple line) in the ribosomal P-site, (ii) Trp-12 (light blue) of the TnaC-tRNAPro peptide (blue line) in the peptide exit tunnel, (iii) proline residue 24 (light yellow) of the TnaC-tRNAPro peptidyl-tRNA, and (iv) free tryptophan (pink) at the peptidyl transferase center. Locations of residues are based on the studies described in this paper and in Cruz-Vera et al. (7). The positions of nucleotides A2058 and A2509, nucleotides whose methylation was affected by antibiotic presence, are shown. An asterisk marks each residue we observed to affect induction, or whose methylation was altered by the presence of tryptophan and/or an antibiotic.
In the present study, our primary objective was to characterize the binding site for free tryptophan in the ribosome-TnaC-tRNAPro complex. We showed that tryptophan affects the reaction of an aminoacyl-tRNA with TnaC-tRNAPro (Fig. 2): it blocks cleavage of TnaC-tRNAPro by puromycin (Fig. 1), and it reduces binding of the antibiotic sparsomycin to ribosome-TnaC-tRNAPro complexes (Fig. 4). We also established that bound tryptophan and features of the nascent TnaC-tRNAPro produce changes in the peptidyl transferase center; methylation of A site nucleotide A2572 was altered (Fig. 5, brown residue).
The replacement of residue A2572 had been shown to have significant affects on peptidyl transferase activity (20). In the crystal structure of the ribosome, A2572 loops out in a helix connecting the A-loop with the peptidyl transferase center (19, 21). This nucleotide had been shown to be cleaved by hydroxyl radicals generated by Fe(II)EDTA tethered to the 5′ end of A site tRNA (22). This indicates that it is close to the aminoacyl-tRNA in the A site. Mutation of A2451 (Fig. 5, blue residue), a nucleotide involved in the peptidyl transferase reaction (20, 24), resulted in dramatic changes in the accessibility of several other residues in the peptidyl transferase center to chemical probes, including U2585 (Fig. 5, yellow residue), U2506, and A2572 (23). Also, the observed changes in the DMS reactivity of nucleotide A2572 are believed to be due to conformational changes in the peptidyl transferase center upon activation of the ribosome (24). Thus, A2572 could be a sensor of conformational changes in the peptidyl transferase center resulting from spatial displacement of several key nucleotides. It is also conceivable that tryptophan interacts directly with A2572 or that it interacts with another nucleotide in the peptidyl transferase center, producing the observed change in methylation of A2572. In either case, a change might occur in the peptidyl transferase center affecting its activity.
Sparsomycin is known to be a very efficient inhibitor of peptide bond formation in ribosomes of Eubacteria, Archaebacteria, and Eukaryotes. Sparsomycin, unlike chloramphenicol, competes directly with puromycin for a common binding site in the ribosomal A site (18). We observed that tryptophan does compete with puromycin or sparsomycin action in ribosomes (Figs. 1 and 4 A). However, tryptophanol, which we suggest also interacts with the tryptophan binding site, does not compete with either antibiotic (Figs. 1 and 4 B). Possibly, the changes or interactions resulting from the presence of tryptophan, but not those produced by tryptophanol, do cause specific changes in the A site domain. Affinity labeling and crosslinking data have demonstrated that sparsomycin appears to bind to nucleotide A2602 in the 23S rRNA peptidyl transferase domain (25). Mutations introducing changes at two other nucleotide positions in the peptidyl transferase center of the ribosome of Halobacterium halobium, C2471 and U2519 [E. coli C2452 (Fig. 5, blue residue) and U2500, respectively], conferred resistance to low concentrations of sparsomycin (26). Also, a sparsomycin-resistant mutant isolated in the archaebacterium Halobacterium salinarium was shown to lack posttranscriptional modification of U2603 [E. coli numbering U2584 (Fig. 5, yellow residue)], a universally conserved residue located within the peptidyl transferase loop of 23S rRNA (27). Each of these nucleotides is potentially a good target for tryptophan binding and/or action.
Our results suggest that the presence of tryptophan affects the locations of several key nucleotides in the peptidyl transferase center. These repositioned nucleotides could be responsible for the inability to hydrolyze TnaC-tRNAPro when factors such as tryptophan are bound at the ribosomal A site. Additional experiments will be required to identify which specific nucleotide or nucleotides are involved in tryptophan action and how they are affected by the presence of Trp-12 of TnaC-tRNAPro in the ribosome exit tunnel.
Materials and Methods
Bacterial Strains and Plasmids.
Two plasmids were used as templates for the synthesis of tnaC biotinylated mRNAs. pGF25-00 has wild-type tnaC, whereas pGF25-14 (tnaCW12R) contains a mutant tnaC in which the Trp-12 codon was changed to an Arg codon. Both of these plasmids also have the wild-type region between tnaC and tnaA genes, followed by the sequence specifying the rpoBC transcription terminator (4). In tryptophan-induction assays performed in vivo, we used E. coli SVS1144 {W3110 bglR551 Δ[lac-argF] U169 (tna p tnaA′-′lacZ)} as the host strain (28). SVS1144 was transformed with plasmids pCV25-00A25 (tnaCAUA25), pCV25-14A25 (tnaCAUA25, W12R), or pCV25-00U25 (tnaCAUU25), prepared by directed mutagenesis of pGF25-00 and pGF25-14, inserting an AUA or AUU codon (a rare and common isoleucine codon, respectively) between the CCA24 and UGA25 codons of tnaC.
Isolation of Biotinylated Ribosome–mRNA Complexes: Methylation-Protection and Antibiotic-Protection Assays.
Isolation of ribosome–mRNA complexes and in vitro translation reactions were performed essentially as described in ref. 4. One milliliter of an in vitro translation reaction performed with biotinylated tnaC mRNA was used to isolate complexes. Streptavidin beads were bound to ribosome–biotinylated mRNA complexes (bead complexes), and the complexes were resuspended in 1 ml of a reaction buffer (35 mM Tris-acetate, pH 8/10 mM Mg acetate/175 mM K glutamate/10 mM ammonium acetate/1 mM DTT/2 mM tryptophan). To eliminate translation termination at the termination codon for tnaC(W12R) when in vitro translation reactions were performed with biotinylated tnaC(W12R) mRNA, cell-free extracts were pretreated with anti-RF2 antibodies, as described in ref. 4, and the bead complexes were resuspended in 0.3 ml of the reaction buffer. Wild-type biotinylated tnaC mRNA was always treated identically, for comparison. Samples of the bead complexes were washed twice with 5 vol of the reaction buffer, in the presence or absence of 2 mM tryptophan, and resuspended in the initial volume of the same buffer used in the wash.
Methylation-protection assays were performed by using 50 μl of the bead complexes after their washing and resuspension. The solutions were mixed and incubated with the various compounds being tested. To methylate 23S rRNA nucleotides, DMS was added to the mixtures, which were incubated as indicated in Fig. 3. The methylation reaction was stopped by adding 25 μl of a solution containing 1 M Tris·HCl (pH 8) and 1.4 M 2-mercaptoethanol. The final reaction was mixed with 325 μl of 1 mM EDTA, total RNA was extracted with phenol chloroform, and the RNA was precipitated by adding 50 μl of 3 M sodium acetate (pH 5), 1 μl of 20 mg/ml glycogen, and 2 vol of ethanol. The pellet was dissolved in 10 μl of water. Approximately 2 μg of total RNA was used in primer-extension analyses by reverse transcription (AMV; Gibco) with 5′-32P-labeled deoxynucleotide primers (29). Primers 5′-TCCGGTCCTCTCGTACT-3′ and 5′-CTATCCTACACTCAAGGCTC-3′, complementary to nucleotides 2654–2674 and 2102–2122 of 23S rRNA, were used to detect modified nucleotides in 23S rRNA.
Puromycin-competition assays were performed with 10 μl of bead complexes, after washing and resuspension. Solutions were mixed with tryptophan (Figs. 1 and 3 C) or tryptophanol (Fig. 3 C). In some instances, complexes were incubated in the presence of puromycin, which was added to induce hydrolysis of the peptidyl-tRNA. The amount of TnaC-tRNAPro remaining after the reaction produced by puromycin was measured by Northern blot assays. Then, the components of the final reaction mixtures were resolved by electrophoresis on 10% Tricine-SDS protein gels and transferred to nitrocellulose membranes, and the membranes were hybridized as described by Varshney et al. (30) using a 5′-32P-labeled deoxynucleotide primer, 5′-CCCTAGTTTAAGGCC-3′, that was complementary to the tRNA2Pro sequence (16).
Detection of TnaC-tRNAProin Vivo.
Cells were cultured in supplemented minimal medium (31) with 100 μg/ml ampicillin in the presence or absence of 100 μg/ml tryptophan. Cultures were harvested in mid-log phase (OD600 = 0.8), washed, and resuspended in a fresh buffer (10 mM NH4Cl/175 mM K acetate/10 mM MgCl 2 /35 mM Tris·HCl, pH 8.0/1 mM DTT) containing 2 mM tryptophan. 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 × g. The resulting extracts were resolved by electrophoresis on 10% Tricine-SDS gels, the bands were transferred to nylon membranes, and the TnaC-tRNAPro band was detected as indicated above (32).
Acknowledgments
We thank Koichi Ito and Yoshikazu Nakamura for providing antibodies to E. coli RF2 and Catherine Squires and Matthew Sachs for their excellent comments on the manuscript. This work was supported by National Science Foundation Grant MCB-0093023 (to C.Y.).
Footnotes
- *To whom correspondence should be addressed. E-mail: yanofsky{at}cmgm.stanford.edu
-
Author contributions: C.Y. designed research; and L.R.C.-V. and M.G. performed research.
-
Conflict of interest statement: No conflicts declared.
- Abbreviations:
- DMS,
- dimethyl sulfate.
- © 2006 by The National Academy of Sciences of the USA
References
Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide
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Image credit: Helena Brendler (The University of British Columbia, Vancouver, Canada).
The health span of supercentenarians might be partly explained by the expansion of T cells that kill infected or abnormal cells.
Image credit: PNAS.
Strict clean air policies implemented during 2013–2017 in China helped decrease the national mean particulate matter exposure and avert an estimated 370,000 deaths annually.
Image credit: Pixabay/ii7017.
A study finds extreme ranges of heart rates in blue whales during diving, feeding, and surfacing.
Image credit: Duke Marine Robotics and Remote Sensing Lab.