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. 2010 Dec 24;285(52):40933-42.
doi: 10.1074/jbc.M110.164152. Epub 2010 Sep 30.

Sequence requirements for ribosome stalling by the arginine attenuator peptide

Christina C Spevak  1 , Ivaylo P Ivanov, Matthew S Sachs
Affiliations

Sequence requirements for ribosome stalling by the arginine attenuator peptide

Christina C Spevak et al. J Biol Chem. .

Abstract

The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9-20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.

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Figures

FIGURE 1.
FIGURE 1.
The 5′ leader region of the arg-2 LUC gene used in this study. The sequence begins with the T7 RNA polymerase-binding site and ends with the LUC coding region. The 24-amino acid sequences of the arg-2 AAP and the N terminus of LUC are indicated. The forward primers (RF1, CS1) and the final reverse primers (RF12, CS12) that were used for PCR are shown by horizontal arrows. One of the reverse primers used to introduce the individual mutations (pCS201) used for the first round of PCR is shown by a horizontal arrow (see supplemental Table 1 for a list of primers used for all individual mutations). The sequence for which the reverse complement was synthesized and used as primer ZW4 for toeprint analysis is indicated by a horizontal arrow below the sequence.
FIGURE 2.
FIGURE 2.
Summary of effects of changing individual amino acids on the function of the AAP. The wild-type arg-2 AAP sequence is shown. Circles indicate residues 6–24 that were changed to Ala, Pro, or the indicated amino acids individually and tested for function in N. crassa translation reaction mixtures containing low Arg (10 μm) versus 500 μm Arg (A) or 2 mm Arg (B). The effects of single amino acid changes in the region important for regulation were assessed by luciferase and toeprint assays. Red indicates regulation is lost by the specific changes; yellow indicates regulation is reduced; green indicates regulation is retained. The red star indicates the D12N mutation, which is the control for lost regulatory function. Triangles indicate mutations tested at 500 μm Arg only in this or other studies.
FIGURE 3.
FIGURE 3.
Toeprint analyses of the critical residues in Arg-specific regulation. The different constructs examined are indicated on the top. The ratio of luciferase activity produced after 30 min in reaction mixtures containing 10 μm Arg versus 500 μm Arg is also indicated at the top. Equal amounts of synthetic RNA transcripts (120 ng) were translated in 20-μl reaction mixtures at 25 °C. Reaction mixtures contained 10 μm (−) or 500 μm (+) Arg and 10 μm each of the other 19 amino acids. After 20 min of translation, 3 μl of translation mixtures were toeprinted with primer ZW4 and analyzed next to dideoxynucleotide sequencing of wild-type construct pR301. −EXT, RNA without extract; −RNA, extract not programmed with RNA.
FIGURE 4.
FIGURE 4.
The constructs used to analyze the minimal sequence required for an internal AAP to function. Deletions from the N- and C-terminal regions are compensated by the LUC sequences on both sides (in lowercase). The LUC residues that are at the same positions as the original AAP residues are capitalized. Luc residues bordering AAP-residues in pCS913 were changed to Ala-residues in pCS914 as indicated. Pulse-chase time course experiments and SDS/PAGE were performed to examine whether the modified internal AAP continued to cause ribosome stalling. Green, Arg-specific stalling observed; red, Arg-specific stalling not observed.
FIGURE 5.
FIGURE 5.
Polypeptide synthesis time course in N. crassa cell-free extracts. To examine the core AAP sequence required for regulation, transcripts specifying some of the AAP constructs depicted in Fig. 4 were translated in extracts in low (10 μm) or high (2000 μm) Arg as indicated. Edeine was added at 2 min (arrow), and 10-μl aliquots of extracts were removed at the indicated time points for analysis by SDS/PAGE. Arrowhead I refers to the intermediate that accumulates when the AAP stalls ribosomes; arrowhead F refers to the full-length polypeptide. The full AAP and Ala-flanked core AAP sequences area is shown below the corresponding panels in capital letters. The flanking Ala residues that align with Ala residues in the full AAP are indicated by capital letters; others are lowercase. A, pCS801; B, pCS907; C, pCS908; D, pCS912; E, pCS909; F, pCS914.
FIGURE 6.
FIGURE 6.
Evolutionary conservation of predicted fungal AAPs. Frequency plot representation of the amino acid conservation of different AAPs found in 5′-UTRs of transcripts specifying the fungal homologs of the small subunit of arginine-specific carbamoyl phosphate synthetase is shown. Letter sizes correspond to the frequency of occurrence of the specified amino acid. Each line represents the conservation of AAPs in a particular taxonomic group named on the right followed in parentheses by the number of different AAP sequences used to generate the line. The sequence of the N. crassa AAP is shown on top, and the numbering of its amino acid residues is indicated. The exact sequence of each AAP used to generate this frequency plot representation is shown in supplemental Fig. 2.
FIGURE 7.
FIGURE 7.
Effects of Arg, RGD, and GRGDS on the AAP. Transcripts encoding either wild-type (WT) or D12N mutant uORF were examined in N. crassa extracts. Reactions were supplemented with different concentrations of Arg, RGD, or GRGDS as shown. The products obtained for primer extension of pure RNA in the absence of translation reaction mixture (−EXT) and from a translation mixture not programmed with RNA (−RNA) are shown for comparison. A, shown is the transcript specifying the wild-type AAP; B, shown is the transcript specifying the D12N AAP. Radiolabeled primer ZW4 (19) was used for primer extension analysis and for sequencing of each ARG2-LUC template (sequencing was accomplished using the Thermo Sequenase Cycle Sequencing kit (United States Biochemical Corp.). The nucleotide complementary to the dideoxynucleotide added to each sequencing reaction is indicated below the corresponding lane (C′, T′, A′, and G′) so that the sequence of the template can be directly deduced; the 5′-to-3′ sequence reads from the top to bottom. The box indicates the uORF termination codon; the toeprint signal ≈13-nucleotides downstream of the stop codon (arrowhead) corresponds to ribosomes with the termination codon in the A site.
FIGURE 8.
FIGURE 8.
Time course of polypeptide synthesis with RGD. A, diagram of the coding region of the Met9-AAP-LUC-AAP-LUC fusion polypeptide analyzed. B, the RGD tripeptide confers regulation when added to translation reactions with N. crassa programmed with the Met9-AAP-LUC-AAP-LUC mRNA (17) and incubated at 25 °C. Reaction mixtures contain 10 μm Arg, 300 μm RGD, or 300 μm Arg as indicated, and 10 μm each of the other amino acids. Edeine was added at 2 min (arrow) and polypeptide products were analyzed by SDS/PAGE following removal of samples at the indicated timepoints. Arrowhead N refers to intermediate Met9-AAP, arrowhead I refers to intermediate corresponding to Met9-AAP-LUC-AAP, and arrowhead F refers to the full-length polypeptide.
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References

    1. Sachs M. S., Geballe A. P. (2006) Genes Dev. 20, 915–921 - PubMed
    1. Morris D. R., Geballe A. P. (2000) Mol. Cell. Biol. 20, 8635–8642 - PMC - PubMed
    1. Hood H. M., Neafsey D. E., Galagan J., Sachs M. S. (2009) Annu. Rev. Microbiol. 63, 385–409 - PubMed
    1. Ramu H., Mankin A., Vazquez-Laslop N. (2009) Mol. Microbiol. 71, 811–824 - PubMed
    1. Ito K., Chiba S., Pogliano K. (2010) Biochem. Biophys. Res. Commun. 393, 1–5 - PMC - PubMed

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