Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Mar 23;101(12):4059-64.
doi: 10.1073/pnas.0400554101. Epub 2004 Mar 12.

A nascent polypeptide domain that can regulate translation elongation

Peng Fang  1 , Christina C Spevak, Cheng Wu, Matthew S Sachs
Affiliations

A nascent polypeptide domain that can regulate translation elongation

Peng Fang et al. Proc Natl Acad Sci U S A. .

Abstract

The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Polypeptide synthesis time course in N. crassa cell-free extracts. (A) The Met9-AAP-globin-AAP-LUC construct used (DNA sequence in Fig. 5A). Asterisks indicate where wild-type AAP Asp codons were changed to Asn. Arrowhead N, the C terminus of the Met9-AAP polypeptide-intermediate; arrowhead I, the C terminus of the Met9-AAP-globin-AAP intermediate; arrowhead F, the C terminus of the completed polypeptide. Unique restriction enzyme sites are indicated. Transcripts specifying Met9-AAPm-LUC-AAPm-LUC (B and C) or Met9-AAPw-LUC-AAPw-LUC (D and E) were translated in extracts in low (B and D) or high (C and E) Arg (see text). 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 (21). Arrowhead N, the intermediate Met9-AAP; arrowhead I, the polypeptide-intermediate Met9-AAP-globin-AAP; arrowhead F, the full-length polypeptide.
Fig. 2.
Fig. 2.
CTAB precipitation of peptidyl tRNA from translation extracts and quantitative analysis of polypeptide intermediates and products. N. crassa extracts (150 μl) were programmed with Met9-AAPw-globin-AAPw-LUC mRNA and incubated with high Arg, as described in Fig. 1. Edeine was added at 2 min and 10-μl aliquots removed at the indicated time points for analyses. (A) Total translation product analysis. (B) CTAB-precipitated translation product analysis. Arrowheads are as in Fig. 1. (C) Quantitative analysis of translation products obtained from data in A and an independent experimental replicate. The radiolabel in bands N, I, and F was determined by using imagequant 5.1 (Molecular Dynamics). The amount of radiolabel in band F at 30 min in each experiment was normalized to 100%, and the radiolabel in each band at each time point was calculated as a fraction of this value. White, radiolabel in intermediate N; gray, radiolabel in intermediate I; black, radiolabel in full-length polypeptide F. The total (not normalized) radiolabel in bands N, I, and F at 3 min was 88% of the amount at 30 min.
Fig. 3.
Fig. 3.
Toeprint analysis of ribosome stalling at AAP domains in Met9-AAP-globin-AAP-LUC mRNA. Separate gels and primers were used for analyses of stalling at the N-terminal (Upper) and internal (Lower) AAP domains for optimal resolution. N-terminal and internal AAP domains are indicated as wild-type (AAPw)or mutated (AAPm) above or below the corresponding lanes. Transcripts were translated in 20-μl reaction mixtures containing 10, 500, 2,000, or 5,000 μM Arg as indicated and 10 μM of the other amino acids and analyzed as described (13, 25). (Left) Sequencing reactions for the Met9-AAPw-globin-AAPw-LUC template. The sequence can be directly read 5′ to 3′ from top to bottom. Controls: Products obtained from primer extension of RNA (18 ng) in the absence of extract (–EXT) and from an extract not programmed with RNA (–RNA). Primers FP94 and FP93 (Fig. 5A) were used for the experiments shown (Upper and Lower, respectively). The open circle indicates the mRNA 5′ end; the arrow indicates the position of ribosomes at the first Met (start) codon. Translation of the nine contiguous Met codons was slow, as evidenced by the toeprints of ribosomes in this region. Asterisks mark each AAP's final GCG codons, which lie ≈16 nt upstream of the toeprints corresponding to the stall sites associated with the production of polypeptide intermediates N and I (indicated); toeprints that represent translational stalling after the Met9-AAP coding region are indicated with a bracket.
Fig. 4.
Fig. 4.
Time course of polypeptide synthesis in wheat germ extracts and reticulocyte lysates. (A) The Met9-AAP-LUC-AAP-LUC construct used (DNA sequence in Fig. 5B). Designations are as in Fig. 1 A except that arrowhead I is the C terminus of the Met9-AAP-LUC-AAP intermediate. (B) Wheat germ extracts (150 μl) or reticulocyte lysates (100 μl) were programmed with the indicated mRNA (W-W, Met9-AAPw-LUC-AAPw-LUC, M-W, Met9-AAPm-LUC-AAPw-LUC; and M-M, Met9-AAPm-LUC-AAPm-LUC) and incubated at 25°C with either 10 μM or 2,000 μM Arg and 10 μM each of the other amino acids. Edeine was added at 2 min, and polypeptide products were analyzed as described in Fig. 1, except that reticulocyte samples were incubated in loading buffer at 25°C for 30 min before SDS/PAGE, and 3 μl of lysate was loaded per lane. F, full-length polypeptide; I, intermediate corresponding to Met9-AAP-LUC-AAP; N, intermediate corresponding to Met9-AAP.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Keenan, R. J., Freymann, D. M., Stroud, R. M. & Walter, P. (2001) Annu. Rev. Biochem. 70, 755–775. - PubMed
    1. Nagai, K., Oubridge, C., Kuglstatter, A., Menichelli, E., Isel, C. & Jovine, L. (2003) EMBO J. 22, 3479–3485. - PMC - PubMed
    1. Lovett, P. S. & Rogers, E. J. (1996) Microbiol. Rev. 60, 366–385. - PMC - PubMed
    1. Tenson, T. & Ehrenberg, M. (2002) Cell 108, 591–594. - PubMed
    1. Geballe, A. P. & Sachs, M. S. (2000) in Translational Control of Gene Expression, eds. Sonenberg, N., Hershey, J. W. B. & Mathews, M. B. (Cold Spring Harbor Lab. Press, Plainview, NY), pp. 595–614.

Publication types

MeSH terms

LinkOut - more resources