Ribosomal Features Essential for tna Operon Induction: Tryptophan Binding at the Peptidyl Transferase Center

In vitro translation assay and isolation of biotinylated mRNA-ribosome complexes.The Escherichia coli K-12 strains and plasmids used in this study are described in Table 1. To prepare S-30 cell extracts, we used E. coli strains derived from strain SR-14 (5), in which plasmid prrnCsacB was replaced with one of the following plasmids: pNK (rrnB operon) (19), pNH153 (rrnB operon with an insertion of an adenine at position 751 in the 23S rRNA gene) (19), pKKU2609C (rrnB operon with a uridine-to-cytosine change at position 2609 in the 23S rRNA gene) (9), pNKA752C (rrnB operon with an adenine-to-cytosine change at position 752 in the 23S rRNA gene), or pNKA752T (rrnB operon with an adenine-to-thymidine change at position 752 in the 23S rRNA gene). S-30 cell extracts were also prepared from wild-type strain W3110 transformed with plasmid pL22K90W (containing an rlpV mutant gene that specifies an L22 protein with a lysine-to-Trp change at position 90) (5). The S-30 extracts were prepared from 3-liter cultures (optical density at 600 nm of 0.7) grown in Luria broth plus 100 μg/ml ampicillin and 2% glucose. The harvested cells were resuspended in 20 ml of a solution containing 50 mM Tris-acetate (pH 8.0), 10 mM magnesium acetate, 175 mM potassium acetate, 10 mM ammonium acetate, 2 mM dithiothreitol (DTT), and 100 μg/ml phenylmethylsulfonyl fluoride and were disrupted using a French press. The debris was separated by centrifugation at 30,000 × g for 30 min, and the supernatant was distributed as small samples within each tube. The aliquots were frozen using dry ice and stored at −70°C. Biotinylated mRNAs were prepared by using DNA fragments obtained by PCR using plasmid pGF25-00 (tnaC mRNA) or pGF25-14 [tnaC(Trp12Arg) mRNA] (13), as described previously by Cruz-Vera et al. (4). The in vitro translation reactions were performed essentially as described previously (13). The mixture (100 μl) contained 40 mM Tris-acetate (pH 8.0), 2 mM DTT, 0.5 mM GTP, 30 mM phosphoenolpyruvate, 0.3 U/ml pyruvate kinase, 2 mM ATP, 3.5% (wt/vol) polyethylene glycol 8000, 20 μg/ml folinic acid, 10 mM ammonium acetate, 175 mM potassium glutamate, 1 mM spermidine, 7.5 mM magnesium acetate, 20 μg/ml tRNA (E. coli mixture), 40 μg/ml mRNA, 0.37 mM of each amino acid except Trp, and either 30 μl of the original S-30 extract or an S-30 preparation previously treated with the antibody anti-release factor 2 (RF2), as described previously (10). When [³⁵S]methionine (20 μCi; 3,000 Ci/mmol) was used to label TnaC, the corresponding nonlabeled amino acid was not added.

Isolation of biotinylated mRNA-ribosome complexes was performed according to the protocol provided by the Promega Co. To purify the biotinylated mRNA complexes using streptavidin paramagnetic beads, 1.5 ml of a suspension of streptavidin beads was washed three times with 1.0 ml of a wash buffer containing 35 mM Tris-acetate (pH 8.0), 1 mM DTT, 10 mM ammonium acetate, 175 mM potassium glutamate, and 10 mM magnesium acetate, and the beads were resuspended in 1.25 ml of this buffer with 2 mM Trp. After performing the in vitro translation reaction, 250 μl of the total reaction mixture was removed, streptavidin beads were added, the suspension was mixed, and the final mixture was incubated for 10 min at room temperature to allow biotin-streptavidin interactions. The streptavidin beads with bound biotinylated mRNA-ribosome complexes were then separated by using an electromagnetic field and were washed once with washing buffer containing 2 mM Trp and twice with only washing buffer. The final bead preparations were resuspended in 100 μl of washing buffer.

Puromycin protection assays.Puromycin competition assays were carried out with 10 μl of bead complexes obtained from in vitro translation reactions performed with [³⁵S]methionine after washing and resuspension. Solutions were mixed with the same volume of water or a 20 mM Trp solution. In some instances, complexes were incubated in the presence of puromycin, which was added to induce the hydrolysis of the peptidyl-tRNA. After the reaction was stopped by adding 10 μl of a solution containing 10 mM Tris (pH 6.8), 10% sodium dodecyl sulfate, and 2 mM DTT to the mixture, the components of the final reaction mixtures were resolved by electrophoresis on 10% Tricine-sodium dodecyl sulfate protein gels. The dried gels were exposed to X-ray films, and the sections of the dried gels that contained TnaC-tRNA^Pro and TnaC were excised. The radioactivity (counts per minute [cpm]) in these bands was measured by using a scintillation counter. The amount of [³⁵S]methionine-TnaC-tRNA^Pro remaining after the reaction with puromycin was determined by dividing the cpm in the band corresponding to TnaC-tRNA^Pro by the combined cpm of the bands corresponding to TnaC-tRNA^Pro and the TnaC peptide.

Footprinting assays.Footprinting assays were performed as described previously (16). The isolated complexes were resuspended in 100 μl of the appropriate buffer and were modified by exposure to 4 μl of dimethyl sulfate (DMS) (1:6 dilution in ethanol) for 10 min at room temperature. DMS reactions were stopped by adding 50 μl of stop buffer (1 M Tris-HCl [pH 8.0], 1 M 2-mercaptoethanol, and 1 mM EDTA) to the reaction mixture. The final reaction mixtures were diluted with a 10 mM EDTA solution to reach a total volume of 500 μl, and the RNA was then extracted by treatment with phenol.

Modification by methylation was monitored by primer extension analyses using avian myeloblastosis virus reverse transcriptase (GIBCO) and 5′-³²P-labeled deoxyoligonucleotide primers (27). Primers 5′-TCCGGTCCTCTCGTACT-3′ and 5′-CTATCCTACACTCAAGGCTC-3′, complementary to nucleotides 2654 to 2674 and 2102 to 2122 of 23S rRNA, were used to detect modified nucleotides in 23S rRNA. The positions of the modified nucleotides were identified by reference to dideoxy sequencing ladders obtained using the fmol@ DNA cycle sequence system kit (Promega). DNA from plasmid pNK that contained the 23S rRNA gene (19) and the same primers were used for primer extension of rRNA genes.

The narrow region of the ribosome exit tunnel is important for Trp-mediated induction of tna operon expression.Previously, we described studies with mutants bearing alterations in 23S rRNA (9, 19) or changes in ribosomal protein L22 that prevent the Trp-mediated induction of tna operon expression (5, 28). Our results suggested that interactions between these residues in the ribosome exit tunnel and Trp12 of TnaC are essential for some event required for induction (5). The location of Trp12 of TnaC-tRNA^Pro in the narrow region of the ribosome exit tunnel (Fig. 1) is based largely on the experimental findings that K11 of TnaC-tRNA^Pro can be cross-linked to 23S rRNA nucleotide A750 and that Trp12 of TnaC-tRNA^Pro protects 23S nucleotide A788 from methylation (5). These results imply that these residues are very near one another in the 50S subunit (5). The mutants examined have single nucleotide changes in 23S rRNA, U2609C (9), or an A insertion at position 751 (+A751) (19) or the single amino acid change K90W in ribosomal protein L22 (5). The locations of these residues in the 50S ribosome subunit of E. coli are shown in Fig. 1. These residues are in the narrow region of the ribosome exit tunnel (26), a region that we believe is adjacent to Trp12 of TnaC-tRNA₂^Pro (5). Also shown in Fig. 1 is nucleotide A752, which is located between nucleotides U2609 and A751. To determine if this nucleotide is also required for Trp-mediated induction, we replaced it with a C or a T. We introduced these changes into a expressible 23S rRNA gene present on a plasmid and transformed this plasmid into a strain with all the chromosomal rrn genes deleted that had an integrated λ tna _p tnaC tnaA′-′lacZ expression construct (5) (Table 1). Expression of tnaA′-′lacZ was determined by measuring β-galactosidase levels during growth in the presence or absence of Trp, as described previously by Cruz-Vera et al. (5). We observed that the ratio of tnaA′-′lacZ expression with Trp to that without Trp, when the wild-type 23S rRNA gene was expressed, was 26 (Table 2). When expression was measured in strains producing 23S rRNA with the change A752C or A752T, the induction ratio was 1.2 (Table 2). These results indicate that the nucleotide at position 752 can also influence tna operon induction.

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FIG. 1.

Model displaying the presumed region of the E. coli 50S ribosomal subunit of the stalled ribosome complex that contains TnaC-tRNA^Pro and free Trp. Shown at the left is the trinucleotide CCA of TnaC-tRNA^Pro, attached to TnaC, in the P site of the peptidyl transferase center. Also displayed is the segment of the TnaC peptide believed to be located in the ribosomal exit tunnel. The TnaC peptide was drawn as an α-helix since polypeptide chains are known to adopt a helical structure in the ribosome exit tunnel (31). The presumed locations of selected ribosomal residues, free Trp, sparsomycin, and puromycin in the 50S ribosome subunit are also indicated. The model is based on the ribosome structure of E. coli determined previously by Schuwirth et al. (26). Shown are the nucleotides (cyan) and amino acid residues (brown) analyzed in this study as well as some of the nucleotides involved in the peptidyl transferase reaction (violet) and the nucleotides affected by sparsomycin interaction (A2058 and A2059). The positions of the antibiotics sparsomycin (yellow) (based on the ribosome structure of H. marismortui) (24) and puromycin (black) (based on the ribosome structure of H. marismortui) (15) as well as the putative location of the bound free Trp (blue) (the exact location has not been demonstrated) are shown in relation to the positions of the P site occupied by the trinucleotide CCA (lavender-blue) and A site (occupied by a puromycin molecule) of the ribosome. Residues in green, Pro24, Asp16, and Trp12, are in the TnaC peptide; they are conserved and have been shown to be essential for tna operon induction (11, 28). The model was prepared using PyMOL software (7).

The narrow region of the ribosome exit tunnel is involved in the Trp inhibition of puromycin hydrolysis of TnaC-tRNA^Pro.To obtain a better understanding of how residue changes in the ribosomal exit tunnel affect Trp-mediated induction, mutationally altered 23S rRNA or the L22 protein was incorporated into ribosomes, and the behavior of these ribosomes was examined in vitro in tna operon induction experiments. Strains in which all the chromosomal rrn genes had been deleted, strains containing a plasmid overproducing a mutant 23S rRNA, were used to produce ribosomes containing only mutated 23S rRNAs (1) (Table 1). To obtain mutant ribosomes with the L22 K90W mutant protein, a plasmid overproducing L22 K90W was introduced into a wild-type strain containing the rrn operon genes and the L22 gene (W3110) (Table 1). Cell extracts (S-30) prepared from these strains were employed in in vitro translation reactions with separately prepared tnaC mRNAs. As shown previously, with wild-type ribosome complexes, the presence of an excess of Trp inhibits RF2-mediated translation termination, resulting in the accumulation of the TnaC-peptidyl-tRNA, TnaC-tRNA^Pro (13). As shown in Fig. 2A, S-30s prepared with ribosomes with the 23S rRNA change A752C, +A751, or U2609C or the L22 change K90W, unlike the S-30 prepared with wild-type ribosomes, were unable to accumulate TnaC-tRNA^Pro in the presence of Trp (Fig. 2, compare lane 2 with lanes 4, 6, 8, and 10). Furthermore, the accumulation of detectable amounts of the TnaC peptide was poor in reactions performed with mutant ribosomes, with or without Trp, and the intensity of the TnaC band was similar to the intensity of the band obtained with wild-type ribosomes in the absence of added Trp (Fig. 2A). A noninducible mutant tnaC(Trp12Arg) mRNA was also used in these in vitro translation reactions (Fig. 2B). Interestingly, expression of this mutant tnaC, in the presence or the absence of Trp, was also poor compared with the expression observed with wild-type tnaC mRNA in the presence of Trp (Fig. 2B, compare lanes 1 and 2 with 4). However, when we performed in vitro translation assays with the corresponding wild-type or mutant RF2-depleted S-30 extracts, we observed higher levels of TnaC-tRNA^Pro and the TnaC peptide in the presence or absence of Trp with either tnaC mRNA or tnaC(Trp12Arg) mRNA (data not shown). These results establish that the mutant ribosomes that we prepared and examined can still translate wild-type tnaC mRNA and the noninducible mutant tnaC(Trp12Arg) mRNA. In addition, we observed similar levels of the background protein produced in all our in vitro translation assays (Fig. 2). Thus, the ability of our mutant ribosomes to translate this other mRNA appears to have been unaffected. These results can be interpreted as follows: mutant ribosomes translate tnaC, translation is terminated, and the TnaC peptide produced, the translating ribosome, and the tnaC mRNA are released. The TnaC peptide and tnaC mRNA are then degraded, as observed with wild-type ribosomes in the absence of added Trp (data not shown). However, with wild-type ribosomes, when Trp is present, some fraction of the TnaC-tRNA^Pro resists hydrolysis, unlike the result observed when Trp is present with mutant ribosomes. Thus, in Trp-induced stalled wild-type ribosomes, both the TnaC peptide and the tnaC mRNA are protected from degradation (data not shown).

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FIG. 2.

Examination of the products generated from the translation of tnaC mRNA variants using different cell extracts in the presence of Trp. (A) In vitro translation reactions performed with tnaC mRNA and cell extracts containing wild-type (wt) or mutant ribosomes. (B) In vitro translation reactions performed with cell extracts containing wild-type ribosomes and tnaC mRNA or noninducible tnaC(Trp12Arg) mRNA. The cell extracts were prepared from the Δ7rrn strain (Table 1) containing the 23S rRNA gene indicated in the figures or were obtained from a wild-type strain containing all the wild-type rrn genes, a copy of the rlpV wild-type gene, and a plasmid with a mutant rplV gene that overproduces the L22 protein with the K90W change (Table 1). The protein products formed in the absence (−) or presence (+) of Trp were labeled by adding ³⁵S-labeled methionine and resolved by electrophoresis on Tris-Tricine polyacrylamide gels. Northern blot assays were performed as described previously by Cruz-Vera et al. (4), and degradation assays using RNase A were performed to verify the identity of TnaC-tRNA^Pro, as described previously by Gong and Yanofsky (13). This experiment was performed four times, with essentially the same results. An asterisk marks the principal background-labeled band that is produced using cell extracts without tnaC mRNA (see lanes 5 and 6 in B).

Trp is known to inhibit the hydrolysis of TnaC-tRNA^Pro by puromycin, an analog of an aminoacyl-tRNA, as well as hydrolysis initiated by RF2 (11). Conceivably, the ribosomal mutations that prevent induction do so by preventing Trp binding at the peptidyl transferase center. Consistent with this explanation, studies with tnaC have shown that puromycin action is inhibited by ribosome-bound Trp (5, 11). We therefore tested whether Trp would block puromycin action with mutant ribosome complexes. To perform these analyses, TnaC-tRNA^Pro-ribosome-mRNA complexes were isolated using streptavidin beads following pretreatment of ribosomal extracts with an anti-RF2 antiserum (see Materials and Methods). This treatment eliminates the RF2 protein, the protein responsible for the cleavage of TnaC-tRNA^Pro in ribosomes in the absence of added Trp (10). Accordingly, TnaC-tRNA^Pro-ribosome-mRNA complexes accumulate in the presence or absence of added Trp (10). Using these anti-RF2-treated wild-type and mutant ribosome complexes, the ability of Trp to protect TnaC-tRNA^Pro from puromycin cleavage was examined. As shown in Fig. 3, when puromycin was added to wild-type ribosome complexes, TnaC-tRNA^Pro was cleaved, and a prominent cleaved TnaC band was observed (compare lane 1 with lane 2 in the wild-type panel). This cleavage was blocked by added Trp (Fig. 3, compare lane 2 with lanes 3 and 4). With all the mutant ribosome complexes examined, however, Trp was incapable of protecting TnaC-tRNA^Pro from hydrolysis by puromycin; consequently, a prominent TnaC band was observed (Fig. 3, compare lane 2 with lanes 3 and 4 in the mutant panels). The TnaC peptide band is prominent in these isolated complexes because it is not degraded to the extent observed following translation termination in in vitro assays using crude, nonisolated complexes (Fig. 2). Accordingly, the levels of TnaC peptide observed with isolated complexes (Fig. 3) are higher than the levels observed in complete S-30 in vitro reactions (Fig. 2). The results in Fig. 3 cannot be due to greater susceptibility of mutant ribosomes to puromycin action since we observed the same TnaC-tRNA^Pro hydrolysis ratio using different concentrations of puromycin with wild-type or mutant ribosomes in the absence of Trp (not shown). Thus, Trp is unable to protect TnaC-tRNA^Pro from puromycin action with complexes formed with specific mutant ribosomes in the absence of RF2. Therefore, either these mutant ribosomes are incapable of binding Trp or, when they bind Trp, this amino acid cannot block puromycin action.

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FIG. 3.

Examination of TnaC-tRNA^Pro hydrolysis by puromycin using isolated mutant and wild-type (wt) ribosome complexes. Isolated ³⁵S-labeled TnaC-tRNA^Pro-ribosome-tnaC mRNA complexes containing the 23S rRNA genes or L22 mutant protein K90W, indicated in the figure, were incubated with different concentrations of Trp and then exposed to puromycin (Puro) action by adding (+) 0.02 mM puromycin to 10 nM complexes. The mixtures were then incubated for 10 min at 37°C, the reaction was stopped, and the labeled products were resolved by electrophoresis on Tris-Tricine polyacrylamide gels. The percentage of TnaC as TnaC-tRNA^Pro was calculated by dividing the cpm of the band corresponding to TnaC-tRNA^Pro by the combined cpm of the bands corresponding to TnaC-tRNA^Pro plus the band corresponding to the TnaC peptide. This experiment was performed two times, with essentially the same result.

Changes in 23S rRNA and the L22 protein prevent Trp from inhibiting sparsomycin binding to the ribosome.In previous studies of induction of the wild-type tna operon, it was shown that Trp can compete with sparsomycin, an antibiotic that interacts with the peptidyl transferase center (15, 20). Trp reduces sparsomycin's ability to increase DMS methylation of nucleotides A2058 and A2059 of 23S rRNA of wild-type ribosomes (Fig. 4A, lanes 3 and 4; also see Fig. 1); the effect on A2059 methylation is more prominent (4). Competition assays were therefore also performed between sparsomycin and Trp using complexes containing mutant ribosomes. Unlike wild-type complexes, complexes with the A752C, +A751, U2609C, or L22 K90W changes were not protected by Trp from sparsomycin-induced RNA methylation (Fig. 4A, compare lane 4 with lanes 6, 8, and 10, and B, compare lane 2 with lane 4). These findings suggest that in complexes containing mutant ribosomes with changes that prevent Trp-mediated induction, Trp cannot compete with sparsomycin. Thus, Trp binding and inhibition of sparsomycin action appear to have been eliminated by these ribosomal mutations.

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FIG. 4.

Footprinting analysis of the effects of residue changes in the peptidyl transferase center upon adding Trp to isolated complexes containing wild-type or mutant ribosomes. Isolated TnaC-tRNA^Pro-ribosome-tnaC mRNA complexes were obtained from in vitro translation reactions performed using cell extracts obtained from Δ7rrn strains that express the 23S RNA genes indicated or using cell extracts obtained from a wild-type (wt) strain producing the wild-type or mutant L22 protein. The isolated complexes were incubated without (−) or with (+) Trp for 10 min at 37°C. A sparsomycin (Sps) solution was (+) or was not (−) added to the reaction mixture, and the mixture was incubated for an additional 10 min. The mixtures were then exposed to DMS for 10 min at room temperature. Total RNA was extracted and used as a template in primer extension assays. (A and B) An oligonucleotide complementary to nucleotides 2102 to 2122 was used in primer extension assays. (C) Primer extension analyses with mutant and wild-type ribosomes were performed to examine Trp protection from DMS methylation of nucleotide A2572. No sparsomycin was added. An oligonucleotide primer complementary to nucleotides 2654 to 2674 was used.

It was shown previously with wild-type complexes (4) that bound Trp reduced the methylation of A2572, a nucleotide located in the vicinity of the peptidyl transferase center (Fig. 1). Trp did not reduce the methylation of A2572 in complexes containing the ribosomal changes examined in the present study to the extent observed with wild-type ribosomes (Fig. 4C, compare lanes 1 and 2 with the remaining lanes). These findings also suggest that Trp is incapable of binding effectively to the mutant ribosome complexes that were examined.