Sequence Requirements for Ribosome Stalling by the Arginine Attenuator Peptide^*

DNA Templates

For scanning mutagenesis to replace AAP residues with Ala, Pro, and selected other residues, synthetic oligonucleotides (Sigma Genosys, The Woodlands, TX) were designed for use in megaprimer PCR (25) to place specific mutations affecting the AAP in PCR-generated DNA fragments that contained BglII and NcoI restriction sites near their 5′ and 3′ ends, respectively (supplemental Table 1). After digestion with these enzymes, the PCR products were ligated to a reporter vector (pR401) for further studies. The PCR template was pR301 (26). Forward primers CS1 (5′-GAGCACCGCCGCCGCAAG-3′) or RF1 (5′-CCGCAAGGAATGGTGCAT-3′) were used for first-round PCR, and the various mutagenic primers named in supplemental Table 1 were used as the reverse primers for first round PCR. The product from the first round PCR (megaprimer) was used as the forward primer, and primers CS12 (5′-CCTTTCTTTATGTTTTTGG-3′) or RF12 (5′-TGTTTTTGGCGTCGGTGA-3′) were used as the reverse primers. The final megaprimer PCR products were digested with BglII and NcoI and ligated to similarly digested and gel-purified vector pR401, which differed from pR301 by its containing a unique (and diagnostic) NdeI site. This vector contains the T7 RNA polymerase promoter and firefly luciferase coding region.

For analyses of the minimal functional AAP domain, AAP sequences were ligated to a vector that was derived from plasmid pLL301 (see the supplemental data in Ref. 17). This vector, pCS801, contains a T7 RNA polymerase promoter to enable in vitro synthesis of a polyadenylated transcript containing a reporter ORF consisting of (i) nine methionine codons at the N terminus, (ii) an HA tag, (iii) a luciferase domain, (iv) a modified AAP region containing strategically placed unique restriction sites to enable mutation or truncation of the AAP region, and (v) another luciferase domain (see Fig. 4). Synthetic complementary oligonucleotides were annealed and then ligated to appropriately digested pCS801 vector to produce plasmids (supplemental Table 2 and Fig. 4) containing mutated and/or truncated AAP coding segments specifying the amino acid sequences depicted in Fig. 4. All mutations were verified by DNA sequencing. Plasmid DNA templates were purified by using either the Plasmid Midi Prep kit (Qiagen) or the Wizard Plus Midi Prep kit (Promega).

RNA Synthesis, Cell-free Translation Reactions, and Primer Extension Inhibition (Toeprint) Assays

Capped, polyadenylated RNA was synthesized with T7 RNA polymerase from plasmid DNA templates that were linearized with EcoRI, and the yield of RNA was quantified (27). The reaction conditions for in vitro translation using N. crassa extracts, the preparation of extracts, and the measurements of luciferase enzyme activity were as described (11, 15, 27, 28). Translation reaction mixtures were incubated at 25 °C for 30 min, and translation was halted by freezing reaction mixtures in liquid nitrogen. For firefly luciferase activity measurements, equal amounts (12 ng) of mRNA were used to program extracts; 2.5 ng of synthetic mRNA encoding Renilla (sea pansy) luciferase was added to all reactions to serve as an internal control (15).

To assess Arg-specific ribosome stalling by monitoring the transient accumulation of stalled intermediates in polypeptide synthesis in response to Arg, the previously described [³⁵S]Met pulse-chase protocol (17) was used with micrococcal nuclease-treated N. crassa extracts or micrococcal nuclease-treated wheat germ extracts (Promega). Translation reaction mixtures (20 μl) were programmed with 60 ng of RNA, and [³⁵S]Met was used at a final concentration of 0.5 μCi/μl. Translation reactions were incubated for 2 min and then edeine was added to block additional rounds of translation initiation.

The primer extension inhibition (toeprint) assays were accomplished using ³²P-labeled primer ZW4 as described (27, 29). The gels were dried and exposed to screens for ∼24 h before analysis with a Molecular Dynamics PhosphorImager. All toeprint data shown for the AAP residue substitutions were representative of two or more independent experiments with two independently prepared RNAs. The effects of RGD and GRGDS on ribosome stalling were representative of two independent experiments with independently prepared RNAs.

Bioinformatic Analysis of AAP Conservation

For comparative sequence assembly and analysis, 76 new AAP sequences were compiled using methodology described previously (30). These sequences and 44 known AAP sequences (8) were compared as described (30). Specific details are indicated in the legends of Fig. 6 and supplemental Fig. 2.

Systematic Mutagenesis Revealed Key Residues in the AAP

Synthesis of the 24-residue evolutionarily conserved AAPs caused ribosomes to stall on the mRNA in response to high Arg concentrations. The amino acid sequence of the AAP, not the nucleotide sequence specifying it, is responsible for regulation (11). To better understand the sequence requirements for AAP-mediated ribosomal stalling, we performed alanine-scanning mutagenesis to determine which residues of the AAP were important for Arg-specific regulation. Proline-scanning mutagenesis was also performed to determine whether substitution of each residue with Pro, which should constrain peptide folding, had different effects on the AAP regulatory function than did Ala.

Because AAP residues 2–5 are entirely dispensable for regulation (11), we started scanning mutagenesis on residue Ser-6 (Figs. 1 and 2). Mutations were placed into the N. crassa AAP coding region (Fig. 1, supplemental Table 1) using megaprimer PCR (25). To assess the effects on uORF constructs containing individual alanine or proline substitutions, the effects of each mutation on regulation were compared with regulation mediated by the wild-type uORF and by a uORF in which Asp-12 was mutated to Asn (D12N). The D12N mutation abolishes regulation in vivo (13) and in vitro (11, 15, 27).

Equal amounts of capped and polyadenylated synthetic RNAs representing each construct were used to program N. crassa extracts. The results of analyzing the effects of adding 10 or 500 μm Arg to translation reaction mixtures programmed with three independently prepared batches of synthetic mRNA are shown in supplemental Table 3. Because the mutated AAPs might be expected to exhibit reduced function and not simply show loss of function, we also compared the effects of two different high Arg concentrations (500 μm or 2 mm) to a low Arg concentration (10 μm) using a fourth independent batch of RNA in a second independent batch of extract (supplemental Table 3 and Fig. 2). The results with different batches of extract and RNA were overall similar with respect to the relative effects of each mutation on regulation.

Examination of the luciferase reporter enzyme activity showed that three fundamentally different effects of mutations could be observed (Fig. 2). The first class of mutations eliminated regulation at either 500 μm Arg (Fig. 2A) or 2000 μm Arg (Fig. 2B). This was observed for Ala and Pro substitutions at residues Asp-12, Tyr-13, Leu-14, and Trp-19, which are each highly conserved in fungi (8). The second class of mutations showed reduced regulation at 500 μm Arg relative to 2 mm Arg. Thus, comparison of Fig. 2, A to B, and analysis of the data in supplemental Table 3 showed that specific mutations at residues including Ser-6, Thr-9, Ser-15, Asp-16, His-17, Leu-18, Arg-20, and Ala-21 resulted in no regulation or reduced regulation in response to 500 μm Arg, but the level of regulation increased when the Arg concentration was raised to 2 mm (i.e. residues marked in red or yellow in Fig. 2A are yellow or green, respectively, in Fig. 2B). The final class of mutations showed no obvious regulatory consequences by this assay (they looked nearly wild type at 500 μm Arg). These substitutions were primarily Ala substitutions near the N and C termini of the AAP (e.g. Val-7, Phe-8, Leu-22, and Asn-23).

The effects of Pro and Ala substitutions on regulation at a specific residue were generally similar, with Pro having a greater impact on regulation in most instances. A case where there was a large difference between Ala and Pro substitution was Ser-10, in which substitution with Pro abolished regulation independent of Arg concentration, but substitution with Ala did not. Interestingly, the highly conserved residue Asp-16 (8) could be substituted with Ala or Pro without eliminating AAP function; however, the D16N mutation eliminated function (Ref. 27 and data not shown).

To determine whether any of the mutations had significant effects on the synthesis of luciferase that were independent of their effects on Arg-specific regulation, we assessed the relative amount of luciferase produced per unit amount of input RNA (data not shown). In all cases luciferase activity was at least 80% that of the wild-type mRNA control in extracts supplemented with 10 μm Arg (low Arg). Thus, none of these mutations appeared to increase stalling in low Arg (i.e. cause constitutive stalling), a result that was borne out by the toeprint assays described next.

To verify that the differences in regulatory effects of these and additional mutations that affected regulation based on luciferase assays arose from differences in the capacity of the specific AAPs to stall ribosomes, the positions of ribosomes on each RNA were assayed by primer-extension inhibition assay (toeprinting) (Fig. 3). In these studies sequencing markers (not shown) were used to identify the positions of toeprint signals corresponding to stalled ribosomes; these signals were absent on toeprint analyses of RNA alone (−EXT, Fig. 3A, lane 5) or extract not programmed with RNA (−RNA, Fig. 3A, lane 6). Data for representative substitutions are shown in Fig. 3. The level of Arg-specific regulation, as determined by luciferase activity measurements in parallel studies, is shown in Fig. 3 above the toeprint data; there is good qualitative correspondence between the extent that Arg caused ribosome stalling at the uORF termination codon and the degree of regulation observed by luciferase assay. As expected (11), in extracts containing high Arg (500 μm) compared with low Arg (10 μm), an increased toeprint signal corresponding to ribosomes stalled at the wild-type uORF termination codon was observed (Fig. 3A, compare lane 2 to lane 1). Also as expected, the D12N mutation did not show an increased toeprint signal at the uORF stop codon in response to high Arg (Fig. 3A, compare lanes 4 and 3). Mutation of residues Asp-12, Tyr-13, or Lys-14 to Ala or Pro abolished regulation, as seen by assay of luciferase activity and by toeprinting (Figs. 2 and 3B). The toeprint results from D16P, D16A, and D16E were also consistent with the luciferase data (Fig. 3C). Substituting the conserved Trp-19 residue with Ala or Pro abolished regulation, but changing it to Tyr did not, as established by luciferase assay and by toeprinting (Figs. 2 and 3D). An alternative aromatic residue substitution, W19F, also did not affect regulation as determined by luciferase assay. These results are consistent with the comparative analysis data across fungi showing that conserved AAPs contained an aromatic residue at this position but not always Trp (Ref. 8 and see below). Additional toeprinting data were obtained for all of the Ala and Pro substitutions shown in Fig. 2; these data (not shown) were also consistent with the luciferase assays.

Because Ser-15, Asp-16, and His-17 are highly conserved in Pezizomycotina and Saccharomycotina (Ref. 8 and see below) and each can be individually changed to Ala with only minimal impact on function in response to 2 mm Arg (Fig. 2B), we substituted Ala for Ser-15, Asp-16, and His-17 in all possible combinations. All three residues could be converted to Ala simultaneously and regulation maintained, albeit at a reduced level. The effects of mutations were additive in that the effects of single mutations generally had less of an impact on regulation than double mutations, which had less impact than the triple mutation (data not shown).

Additional studies were performed to investigate whether altering the distance between the conserved residues Asp-12/Tyr-13 and the conserved aromatic residue Trp-19 affected regulation. Insertion of a single Ala residue after Lys-14 or His-17 or deletion of residue Lys-14 or Leu-18 abolished regulation as determined by luciferase assays (data not shown).

Determination of the Minimal AAP Sequence Required for Regulation

What is the minimal functional domain of the AAP? Previous studies in which the AAP was specified as an independent uORF showed that the N-terminal segment (residues 2–5) of the AAP could be deleted without affecting regulation, but further deletions eliminated regulation (11). Shortening the C terminus significantly impacted regulation (24). It was not established from these studies whether the loss of regulation occurred because the length of the nascent peptide was crucial or specific residues were crucial. Because the AAP (residues 2–24) could be placed internally within a larger polypeptide and the AAP function to stall ribosomes was retained using ribosomes from fungal, plant, and animal sources (17), we adapted this approach to determine the shortest contiguous region of the AAP that could retain stalling function.

We examined the minimal sequences required for the AAP using constructs to systematically remove the N- and/or C-terminal residues of the AAP when it was positioned internally in a larger polypeptide. The effects of removing these residues were assessed by [³⁵S]Met pulse-chase experiments in which a stall in translation is detected by the appearance of a polypeptide intermediate in SDS-PAGE analyses (17). Synthetic mRNAs were derived from plasmid pLL301 (17) and were prepared as described under “Experimental Procedures.” These synthetic mRNAs were used to program cell-free translation extracts containing Arg at low (10 μm) or high (2 mm) concentrations and the other amino acids and [³⁵S]Met at fixed concentrations. A radiolabel was incorporated into these polypeptides essentially only at the nine Met residues at their N termini (some of the constructs used also contained the first Met-codon of the AAP at an internal position). For pulse-chase analyses, translation reactions were incubated for 2 min, then edeine was added to block subsequent rounds of translation initiation, and polypeptide chain elongation progress was monitored by SDS-PAGE (17).

When the entire wild-type AAP is specified as an internal domain (pCS801, Fig. 4), Arg-specific stalling is observed (Fig. 5). A transient intermediate product (labeled I in Fig. 5) is stabilized when the concentration of Arg is high. The results of testing constructs containing various N- and C-terminal systematic deletions of the AAP and examining the radiolabeled translation products using the pulse-chase protocol established the minimal sequence required for Arg-specific regulation are summarized in Fig. 4. Systematic deletion from the N terminus showed that deletion of residues 2–8 (ΔNGRPSVF) did not affect regulation as seen by the presence of the intermediate (I) when Arg concentrations were high, similar to the situation of the full-length AAP (Fig. 5, compare panel A to panel B). Removal of residues 2–9 affected regulation (ΔNGRPSVFT) as no visible intermediate (I) product in the presence of high Arg concentrations was observed (Fig. 5, compare panel C with B and A). Similarly, systematic deletions from the C terminus showed that residues 21–24 (ΔALNA) could be deleted without eliminating regulation (Fig. 5D), whereas deletion of codons 20–24 (ΔRALNA) considerably diminished regulation (Fig. 5E).

After determining the essential residues for Arg-specific regulation by systematically deleting the AAP C and N termini, a construct was created that combined the largest functional deletions from each terminus to produce a construct encoding only 12 of the AAP residues (Thr-9—Arg-20). To eliminate the possibility that residues from the adjacent luciferase domains had compensated or influenced the deleted AAP residues, alanine was substituted for all amino acid positions of the AAP that were shown to be dispensable for function (pCS914, Fig. 4). This transcript was used to program N. crassa and wheat germ extracts, and polypeptide chain elongation was monitored by pulse-chase analysis (Fig. 5F and supplemental Fig. 1). An intermediate (I) corresponding to polypeptide synthesis stalled at the internal AAP was observed to be enhanced in the presence of high Arg in both systems. Thus, pulse-chase analysis showed that the 12 core AAP residues (Thr-9—Arg-20) were sufficient to cause ribosome stalling in response to Arg.

The Ala replacement construct, pCS914, shares two residues within the full-length AAP outside the core (residues Ala-21 and Ala-24). The construct pCS913 (Fig. 4), in which the 12 core AAP residues were bounded by adjacent LUC residues, which eliminated any similar residues with the wild-type AAP, also stalled ribosomes in response to Arg (data not shown), providing evidence that these Ala residues were not crucial for stalling.

The Minimal Functional Domain of the AAP Is Functionally Important in Vivo Based on Evolutionary Conservation

We investigated how well residues identified as important for AAP-mediated regulation in the in vitro experiments correlated with their phylogenetic conservation by conducting an exhaustive comparative sequence analysis. For this purpose a total of 120 AAP sequences were compiled (Fig. 6 and supplemental Fig. 2) including 44 previously described sequences (8). AAP sequences were identified in six fungal phyla: Ascomycota, Basidiomycota, Zygomycota, Blastocladiomycota, Chytridiomycota, and Neocallimastigomycota. A phylogenetic tree of relatedness of these fungi based solely on AAP sequences was highly consistent with current understanding of the evolution of fungi at the level of phyla and subphyla (31, 32). Only two residues, Asp-12 and Tyr-13, are absolutely conserved in all AAPs; the remainder showed varying degrees of conservation. The strict conservation of Asp-12 is consistent with data from the current and previous studies showing its importance for all aspects of AAP-mediated regulation. Similarly, the in vitro results presented here for Tyr-13 show that mutating it to Ala or Pro completely abolished regulation by Arg. The only other residues showing conservation across all taxonomic groups with identifiable AAPs are Lys-14 and Trp-19. The position corresponding to N. crassa Lys-14 is Leu or Ile in all but one of the compiled sequences (in which it is Phe). The position equivalent to N. crassa Trp-19 is Trp in 100 of the 120 AAPs examined and is either Phe or Tyr in the others; thus, it is always an aromatic residue. These conservation data are also consistent with the in vitro data reported here. Five other positions corresponding to N. crassa Ser-10, Ser-15, Asp-16, Leu-18 and Arg-20 are highly conserved in that they contain similar if not identical residues in at least 4 of the 6 fungal phyla for which AAP sequences were identified. Alanine and proline scanning indicated that mutations in these positions had a mild to severe effect on Arg-specific regulation of translation in vitro. Overall, 9 of the 11 residues between Ser-10 and Arg-20 are well conserved across most taxonomic groups, and no residue outside of that region shows similar conservation. All of the well conserved residues lie in a region that coincides almost perfectly with the Thr-9—Arg-20 region identified here through in vitro studies as the minimal contiguous functional domain of the N. crassa AAP. In addition, although the first residue of this domain, Thr-9, is not conserved across different fungal phyla, it is conserved in 99% (110/111) of AAPs from the subkingdom Dikarya, of which N. crassa is a member. Finally, even though many single codon insertions or deletions are present near the N termini of these AAPs and also several insertions and one deletion are seen near the C termini, not a single insertion or deletion occurs between Asp-12 and Trp-19, consistent with the in vitro data showing that insertions/deletions in this region abolished regulation.

A comparative sequence analysis of the different AAPs also revealed significant variation in the identity of the amino acids near the N termini and C termini, but although the N-terminal region also varied considerably in size, the C-terminal region generally did not. The initiation codon of the AAP could be as close as two codons from the position corresponding to N. crassa T9 (the beginning of the minimal contiguous functional domain) or as far as 11 codons. By contrast, although poorly conserved at the amino acid level, the C termini of AAPs usually end a strict distance from the boundary of the minimal contiguous functional domain. In 84% of all AAPs (101/120) the stop codon of the open reading frame is exactly the same distance from Trp-19 codon as it is in the AAP of N. crassa. In another 16 AAPs, the C terminus is extended by one residue, and in a single case, it is extended by two residues. uORF-encoded AAPs extended by one or two residues still function to stall ribosomes in vitro at the uORF stop codon (11). In the case of Pichia pastoris, the stop codon is one position closer to the minimal contiguous functional domain. The AAP in this yeast, at 18 amino acids long, is also the shortest of them all. Based on evolutionary data, it may not have full function because the other sequenced species of the genus Pichia, for example Pichia stipitis and Pichia guilliermondii, lack an AAP-encoding uORF 5′ of the coding sequence of their genes specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. Trichophyton tonsurans presents another curious deviation. In this species the AAP is fused directly in-frame with the main open reading frame. Fusions of the AAP are known to be functional in vitro (11, 17) and in vivo (19).

Arginine Analogs and Arg-containing Short Peptides Provide Insights into the Structural Requirements for Regulatory Function

Because previous studies showed that the level of aminoacylated Arg-tRNA is not important for regulation (16), Arg or a metabolite of Arg must be important. Many Arg analogs have been previously tested for their effects on regulation on the wild-type AAP, such as arginine methyl ester, which conferred regulation, and precursors such as citrulline that did not confer regulation (24). In a pilot study we tested in parallel the Arg-related compounds d-arginine, arginine methyl ester, citrulline, agmatine, argininamide, and l-NG-monomethyl arginine with each of the Ala- and Pro-scanning mutants from Fig. 2 as well as with the wild-type and D12N AAP controls. The results indicated that Arg-methyl ester was the only analog that showed regulation similar to Arg when an equal amount of each RNA were translated in the reaction mixtures supplemented with 500 μm concentrations of each of the Arg analogs (data not shown). This indicated that changing the primary amino acid sequence of the AAP did not alter the responses to these molecules that were structurally related to Arg, and these studies were not pursued further. The results from these and previous studies suggest that the chiral center, guanidino group, and primary amino group of arginine are most important for regulatory function as changing these groups abolished regulation.

Changes at the carboxyl group of Arg appeared to have little effect on regulatory function. This led us to investigate the effects of short peptides containing an Arg residue such as RGD, RGDS, GRGD, and GRGDS (purified peptides from Bachem and Calbiochem). The prediction was that only peptides containing an N-terminal Arg would confer regulation.

RGD and RGDS showed similar effects as Arg in causing Arg-specific negative regulation through the wild-type arg-2 AAP but not the D12N AAP in N. crassa extracts as determined by luciferase assays (data not shown). Examination of the regulatory response at the molecular level by mapping the positions of ribosomes on the transcript by the toeprint assay showed that in the wild-type AAP construct (Fig. 7A), increasing the concentration of Arg from 10 to 100–500 μm in N. crassa cell-free translation reactions resulted in an increased toeprint at the uORF termination codon, which is diagnostic for Arg-specific regulation (Fig. 7A, compare lane 1 to lanes 2–6). Adding the tripeptide RGD at concentrations of 100–500 μm had an effect similar to Arg (Fig. 7A, compare lanes 2–6 to lanes 7–11) on ribosome stalling at the uORF termination codon. However, the addition of 100–500 μm GRGDS to translation reactions containing the wild-type AAP did not result in an enhanced toeprint signal at the termination codon (Fig. 7A, compare lane 1 to lanes 12–16). GRG tripeptide also did not confer regulation.³ In the D12N AAP construct (Fig. 7B), similarly increasing concentrations of Arg, RGD, and GRGDS did not result in an increased toeprint signal at the uORF termination codon (Fig. 7B), consistent with the inability of the D12N AAP to confer regulation.

Was the effect of RGD a consequence of hydrolysis of the tripeptide to release Arg during the course of the reaction to Arg? We added the aminopeptidase inhibitors bestatin and amastatin (33, 34) to reaction mixtures; these additions did not change the regulatory results. Furthermore, analyses of the kinetics of regulation and the effects of adding different concentrations of Arg or RGD to N. crassa extracts showed that both Arg and RGD acted at similar rates on the wild-type AAP-LUC fusion. For example, we performed pulse-chase experiments and detected a similar stalling response to the addition of either 300 μm Arg or 300 μm RGD soon after initiating translation. We programmed extracts with mRNA specifying a fusion protein (Fig. 8A) containing two wild-type AAP domains (pLL301, Ref. 17). We used 300 μm instead of 2 mm concentrations to demonstrate the similar sensitivity of stalling to Arg and RGD at relatively low concentrations. The results (Fig. 8B) show that, in contrast to translation reactions containing 10 μm Arg, adding 300 μm RGD causes the appearance of transient species representing translational stalling at the N-terminal AAP (N) and the internal AAP (I) with kinetics similar to those as a consequence of adding 300 μm Arg. These data indicate that RGD can replace Arg in vitro to stall ribosomes.