Instruction of Translating Ribosome by Nascent Peptide

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Science  13 Sep 2002:
Vol. 297, Issue 5588, pp. 1864-1867
DOI: 10.1126/science.1073997


Expression of the tryptophanase operon ofEscherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. An induction site activated by l-tryptophan is created in the translating ribosome during synthesis of TnaC, the 24-residue leader peptide. Replacing the tnaC stop codon with a tryptophan codon allows tryptophan-charged tryptophan transfer RNA to substitute for tryptophan as inducer. This suggests that the ribosomal A site occupied by the tryptophanyl moiety of the charged transfer RNA is the site of induction. The location of tryptophan-12 of nascent TnaC in the peptide exit tunnel was crucial for induction. These results show that a nascent peptide sequence can influence translation continuation and termination within a translating ribosome.

Recent structural studies with bacterial ribosomes have revealed the features responsible for catalysis of protein synthesis and for antibiotic action. The ribosomal locations of the E, P, and A sites; template RNA; the decoding center; the exit tunnel for the nascent peptide; and the peptidyltransferase center have all been determined (1–4). The exit tunnel (5), for example, passes through the middle of the 50S subunit and is paved mainly by RNA loops (1). Certain nascent peptides when within the peptide exit tunnel can act in cis to alter the progress of translation (6–8). TnaC, the nascent leader peptide of the tryptophanase (tna) operon of E. coli, falls in this group.

Tryptophanase is a catabolic enzyme that degrades tryptophan to indole, pyruvate, and ammonia, allowing tryptophan to serve as a carbon or nitrogen source (9). The tna operon of E. coli consists of a 319–base pair transcribed leader regulatory region, containing a coding region, tnaC, for the 24-residue leader peptide. This leader region is followed by two structural genes,tnaA and tnaB, that encode tryptophanase and a tryptophan permease (10). Initiation of transcription of the operon is regulated by catabolite repression (11). Once initiated, transcription of the structural genes of the operon is subject to Rho factor–mediated transcription termination at transcription pause sites located immediately after tnaC(12). Rho action is prevented by high concentrations of tryptophan.

Tryptophan induction requires synthesis of TnaC, with its crucial tryptophan residue at position 12 (13,14). Recent in vitro studies have shown that induction is due to tryptophan inhibition of release factor 2 (RF2) action at the tnaC stop codon (15). The nascent TnaC-peptidyl-tRNAPro remains uncleaved; its retention stalls the translating ribosome at the tnaC stop codon (15). This blocks Rho factor's access to its binding site located adjacent to the tnaC stop codon, thereby preventing transcription termination (16). Inducing amounts of tryptophan inhibit both TnaC-peptidyl-tRNAProcleavage and TnaC transfer to puromycin (17). Previous studies did not detect the recognition site for tryptophan in the translating ribosome. Here we identify the likely induction site by showing that the tryptophanyl moiety of tryptophanyl-tRNATrp can replace tryptophan as inducer. We also demonstrate the importance of the location of Trp12 of nascent TnaC-peptidyl-tRNAPro in the peptide exit tunnel.

We reasoned that the most likely site of tryptophan binding/induction in the translating ribosome was the region of the ribosomal A site that accepts the incoming aminoacyl-tRNA. The following observations are consistent with this assumption. A specific DNA template was constructed (W25UGA) in which the tnaC stop codon, UGA, was replaced by a tryptophan codon, UGG; this tryptophan codon was followed by a UGA stop codon. This template was examined with standard S-30 preparations (18). We expected that, if the template's coding region was translated to completion, TnaC would have an additional residue, tryptophan, at position 25. However, if the tryptophanyl moiety of tryptophanyl-tRNATrp replaced tryptophan as inducer, the 24-residue TnaC peptidyl-tRNAProwould accumulate in the translating ribosome in the absence of added tryptophan. We observed accumulation of [35S]methionine-labeled TnaC peptidyl-tRNAPro(19) in the absence and presence of added tryptophan (Fig. 1, W25UGA) (20). With the wild-type (WT) template (P24UGA), TnaC-peptidyl-tRNAPro accumulation occurred only in the presence of added tryptophan (Fig. 1A, WT); that is, in the absence of added tryptophan TnaC-peptidyl-tRNAPro was cleaved, producing free TnaC (15). Placing 12 codons between the W25 UGG codon and the UGA stop codon (Fig. 1A, W25+12AA) also led to TnaC peptidyl-tRNAPro accumulation in the absence of added tryptophan. These results show that tryptophanyl-tRNATrpcan inhibit TnaC-peptidyl-tRNAPro elongation, in agreement with our previous finding that tryptophan inhibits TnaC-peptidyl transfer to puromycin (17). A template bearing a proline codon after the codon for W25 behaved like the W25UGA template (Fig. 1A, W25P26UGA). In contrast, inserting a proline codon between the codons for P24 and W25 eliminated peptidyl-tRNA accumulation (Fig. 1A, P25W26P27UGA). Replacing the codon for P24 with a tryptophan codon (Fig. 1A, W24UGA) also eliminated peptidyl-tRNA accumulation.

Figure 1

(A) Trp-tRNATrp–induced TnaC-peptidyl-tRNAPro accumulation. S-30 reaction mixtures were programmed with circularized DNA templates bearing the changes indicated (15). Incubation was in the absence (−) or presence (+) of 1 mM tryptophan. +12AA templates encode a 12-residue segment (MHTQKPTLELLT) added to the COOH terminus of an existing peptide. Radiolabeled products were separated by SDS-PAGE (15). Accumulated [35S]methionine- labeled peptidyl-tRNA is shown. (B) RT-PCR identification of the tRNA of the accumulated peptidyl-tRNA. Oligonucleotide (oligo) pairs specific for tRNA2 Pro and tRNATrp were used in RT-PCR, as indicated. Lanes 1 and 6, E. coli total tRNA was used as template (positive controls). The peptidyl-tRNA in (A) was recovered from each lane in the gel and used as RT-PCR template (15). Lanes 2 and 7, peptidyl-tRNA from reaction W25UGA +Trp was used as template; lanes 3 and 8, that from reaction W25UGA −Trp was used. Lanes 4 and 9, peptidyl-tRNA from reaction W25+12AA +Trp was used as template; lanes 5 and 10, that from reaction W25+12AA −Trp was used.

We identified the tRNAs associated with the accumulated peptidyl-tRNAs with appropriate primers and reverse transcriptase–polymerase chain reaction (RT-PCR) (15). The peptidyl-tRNA accumulated with templates TnaCW25UGA (−/+Trp) and TnaCW25+12AA (−/+Trp) (Fig. 1A) was tRNAPro, not tRNATrp (Fig. 1B). Thus, the presence of Trp-tRNATrp in the ribosomal A site during attempted translation of tryptophan codon 25 inhibits elongation of TnaC beyond proline codon 24.

Inhibition of peptidyl transfer by Trp-tRNATrpdepended on features of the nascent TnaC peptide; for example, replacing the W12 codon of tnaC with an arginine codon eliminated peptidyl-tRNA accumulation (Fig. 1A, R12W25UGA, R12W25+12AA). Moreover, Trp-tRNATrp did not promote peptidyl-tRNA accumulation when proline codon 24 was deleted (Fig. 1A, W24UGA). Because Trp-tRNATrp was also inactive with the WT template, in which codon 25 of tnaC is a stop codon, UGA (Fig. 1A, WT), it appears that a tryptophan codon is required at position 25 for Trp-tRNATrp to function as inducer. Introducing a phenylalanine (Fig. 1A, F25UGA) or a methionine (Fig. 1A, P24+12AA) codon at position 25 did not lead to peptidyl-tRNA accumulation, with or without added tryptophan. Excess phenylalanine was present in the reaction mixture. These findings suggest that after synthesis of TnaC-peptidyl-tRNAPro with W25UGA and W25+12AA templates, a tryptophan codon and Trp-tRNATrp must occupy the A site of the translating ribosome for Trp-tRNATrp to inhibit peptide elongation.

Addition of tryptophan to S-30 reaction mixtures with templates that contained a tryptophan codon at position 25 increased TnaC-peptidyl-tRNAPro accumulation slightly, as did addition of 1-methyltryptophan (1MT), an analog of tryptophan that is an effective inducer both in vivo and in vitro (Fig. 1A) (15). 1MT is at best poorly aminoacylated onto tRNATrp (21); therefore, uncharged 1MT must serve as inducer. Added tryptophan had little or no effect on TnaC-peptidyl-tRNA accumulation with templates F25UGA, P24+12AA, and P25W26P27UGA (Fig. 1A). These findings suggest that, although synthesis of TnaC-peptidyl-tRNAPro may create a tryptophan induction site in the translating ribosome, entry of a tRNA other than Trp-tRNA into the ribosomal A site can compete with tryptophan and prevent induction.

We determined the stability of accumulated TnaC-peptidyl-tRNAPro by measuring its decay after cessation of synthesis. We added kasugamycin to inhibit translation initiation, and we added unlabeled methionine to dilute the labeled methionine. We used WT or W25+12AA templates to direct peptide synthesis in S-30 preparations incubated with and without tryptophan (Fig. 2). With the WT template, we detected TnaC-peptidyl-tRNAPro accumulation only with added tryptophan; upon continued incubation, this peptidyl-tRNA disappeared (Fig. 2A, rows 1 and 2; Fig. 2B.) Addition of chloramphenicol to inhibit peptidyltransferase activity stabilized the peptidyl-tRNA (row 3), whereas adding increasing amounts of RF2 decreased its stability (Fig. 2A, rows 4 and 5; Fig. 2B). With the W25+12AA template, TnaC-peptidyl-tRNAPro was more labile (Fig. 2A, rows 6 and 7; Fig. 2B). Tryptophan addition may have increased the initial amount of this peptidyl-tRNA or delayed its decay slightly; we cannot distinguish between these possibilities.

Figure 2

(A) Decay of accumulated [35S]methionine-labeled TnaC-tRNAPro. Rows 1 to 5: S-30 reaction mixtures ([35S]methionine-labeled products) with the WT tnaC template were incubated without (row 2) or with (rows 1, 3, 4, and 5) 1 mM tryptophan at 37°C for 15 min, and then 1 mM unlabeled methionine and kasugamycin (200 μg/ml) were added. Reaction mixtures without added tryptophan were subjected to time course analysis directly. A reaction mixture with tryptophan was distributed into four tubes, and immediately H2O (row 1, control), chloramphenicol (Cm 500 μg/ml, row 3), or RF2 (rows 4 and 5) was added. Time course samples were taken as indicated. Rows 6 and 7, an S-30 reaction mixture directed by the W25+12AA template was incubated at 37°C for 15 min without tryptophan. After addition of 1 mM unlabeled methionine and kasugamycin (200 μg/ml), one half of each reaction mixture was mixed with tryptophan (1 mM, row 6); the other half was mixed with the same volume of water (row 5). Time course samples were taken as indicated. (B) TnaC-peptidyl-tRNAPro decay curves based on the data shown in (A). No line is shown for row 2.

On the basis of these results and previous data (14–16), tryptophan residue 12 of TnaC appears to be essential for creating the tryptophan induction site in the translating ribosome. To further explore the features of TnaC required for induction, we introduced additional changes in TnaC templates (Table 1). The crucial role of W12 in permitting induction was confirmed; the W12R alteration eliminated induction. The NH2-terminal portion of TnaC was relatively unimportant; additions, deletions, and replacements in this region had no significant effect on peptidyl-tRNAProaccumulation with added tryptophan. In contrast, features of the COOH-terminal portion of TnaC were essential. P24 could not be deleted or replaced (ΔP24, ΔR23P24, P24A, P24S); however, K18 could be replaced by arginine (K18R) without affecting tryptophan induction. The spacing between W12 and P24 appeared to be crucial; insertions, duplications, and deletions decreased or abolished the effect of added tryptophan. The presence of the stop codon UGA after COOH-terminal residue P24 gave the greatest sensitivity to added tryptophan. This observation is consistent with the conclusion that termination is a slower process than elongation (22); thus, RF2 may be less effective than most tRNAs in competing with tryptophan in the A site of the ribosome. These findings suggest that the translating ribosome must recognize the crucial residue W12 at a specific location in the exit tunnel for TnaC-peptidyl-tRNAPro to create an effective tryptophan induction site.

Table 1

Features of the TnaC peptide required for cis action. Circularized DNA templates bearing the changes responsible for the amino acid alterations listed below were tested in S-30 reaction mixtures with or without tryptophan, as described in Fig. 1A, and the radiolabeled products were separated by SDS-PAGE and quantified (15). The amount of [35S]methionine-labeled TnaC-tRNA indicated is relative to that obtained with a WTtnaC template in the presence of 1 mM Trp (+++++). Scoring ranged from no accumulation (−) to strong accumulation (+++++). Δ = deletion; Ins = insertion.

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We believe nascent TnaC-peptidyl-tRNAPro creates anl-tryptophan–specific induction site in the ribosomal A site. Bound tryptophan apparently rearranges or displaces some element of the ribosome essential for catalysis of peptidyl transfer and peptidyl-tRNA cleavage. This conclusion is supported by our finding that Trp-tRNATrp inhibits aminoacyl-tRNA addition to TnaC-peptidyl-tRNAPro. Moreover, Trp-tRNATrp is active as an inducer at very low concentrations (∼0.1 μM) relative to the tryptophan concentration required for induction (>100 μM) (15). The tryptophanyl moiety of bound Trp-tRNATrp presumably occupies the tryptophan induction site (14). However, it is likely that elongation factor Tu, guanosine triphosphate, and/or the associated tRNATrpcontribute to Trp-tRNATrp binding in the ribosomal A site; this would account for the effectiveness of Trp-tRNATrp at lower concentrations than free tryptophan. The induction site could be a newly created site that specifically recognizes tryptophan. Alternatively, the peptidyltransferase center could be displaced or altered during synthesis of TnaC-peptidyl-tRNAPro; thus, when tryptophan enters the center, peptidyltransferase activity is inhibited. It is unlikely that the induction site is formed in the ribosomal D site, a newly identified site proposed to participate in the initial step in decoding (23). Our findings strongly suggest that information inherent in the sequence of the nascent TnaC peptide chain is communicated to the translating ribosome and that this information is used to mediate a response to tryptophan binding at the ribosomal A site. Peptide-ribosome interactions of this type could regulate the rate of peptide chain elongation, facilitate cotranslational protein folding (24), or, as in thetna operon, allow tryptophan to compete with a release factor and force ribosome stalling at a transcript site required for Rho factor binding.

Our findings and those of other investigators therefore attribute to the translating ribosome the ability to sense features of a nascent peptide and of responding by altering one or more events in ribosome action (Fig. 3). Inhibition of peptidyl-tRNA transfer or cleavage may also occur during translation of the uORF (upstream open reading frame) preceding an arg gene of fungi (25), the uORF2 preceding a gene of the human cytomegalovirus (26), and the uORF preceding the coding region for mammalian S-adenosylmethionine decarboxylase (27). Nascent peptides have also been described that inhibit translation elongation (6,28, 29). These examples illustrate ribosomal versatility in mediating regulatory decisions.

Figure 3

Schematic representation of the 50S ribosomal subunit with TnaC-peptidyl-tRNAPro in the P site and a decoding Trp-tRNATrp in the A site. A segment of the peptidyl portion of TnaC-peptidyl-tRNAPro and the tryptophanyl moiety of Trp-tRNATrp are placed in the peptidyltransferase center. We assume the narrowest part of the exit tunnel formed with ribosomal proteins L4 and L22 (1, 28,30) responds to the segment of TnaC containing the crucial residue W12 (enlarged circle) by altering features of the peptidyltransferase center, creating a tryptophan induction site.

  • * To whom correspondence should be addressed. E-mail: yanofsky{at}


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