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. 1997 Sep;17(9):4904–4913. doi: 10.1128/mcb.17.9.4904

Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa.

Z Wang 1, M S Sachs 1
PMCID: PMC232343  PMID: 9271370

Abstract

The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon.

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Selected References

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  1. Anthony D. D., Merrick W. C. Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J Biol Chem. 1992 Jan 25;267(3):1554–1562. [PubMed] [Google Scholar]
  2. Brown C. M., McCaughan K. K., Tate W. P. Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination. Nucleic Acids Res. 1993 May 11;21(9):2109–2115. doi: 10.1093/nar/21.9.2109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cao J., Geballe A. P. Coding sequence-dependent ribosomal arrest at termination of translation. Mol Cell Biol. 1996 Feb;16(2):603–608. doi: 10.1128/mcb.16.2.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao J., Geballe A. P. Inhibition of nascent-peptide release at translation termination. Mol Cell Biol. 1996 Dec;16(12):7109–7114. doi: 10.1128/mcb.16.12.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davis R. H. Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol Rev. 1986 Sep;50(3):280–313. doi: 10.1128/mr.50.3.280-313.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis R. H., Ristow J. L. Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine. J Biol Chem. 1987 May 25;262(15):7109–7117. [PubMed] [Google Scholar]
  7. Delbecq P., Werner M., Feller A., Filipkowski R. K., Messenguy F., Piérard A. A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript. Mol Cell Biol. 1994 Apr;14(4):2378–2390. doi: 10.1128/mcb.14.4.2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doohan J. P., Samuel C. E. Biosynthesis of reovirus-specified polypeptides. Analysis of ribosome pausing during translation of reovirus S1 and S4 mRNAs in virus-infected and vector-transfected cells. J Biol Chem. 1993 Aug 25;268(24):18313–18320. [PubMed] [Google Scholar]
  9. Doohan J. P., Samuel C. E. Biosynthesis of reovirus-specified polypeptides: ribosome pausing during the translation of reovirus S1 mRNA. Virology. 1992 Feb;186(2):409–425. doi: 10.1016/0042-6822(92)90006-b. [DOI] [PubMed] [Google Scholar]
  10. Freitag M., Dighde N., Sachs M. S. A UV-induced mutation in neurospora that affects translational regulation in response to arginine. Genetics. 1996 Jan;142(1):117–127. doi: 10.1093/genetics/142.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geballe A. P., Morris D. R. Initiation codons within 5'-leaders of mRNAs as regulators of translation. Trends Biochem Sci. 1994 Apr;19(4):159–164. doi: 10.1016/0968-0004(94)90277-1. [DOI] [PubMed] [Google Scholar]
  12. Harigai M., Miyashita T., Hanada M., Reed J. C. A cis-acting element in the BCL-2 gene controls expression through translational mechanisms. Oncogene. 1996 Mar 21;12(6):1369–1374. [PubMed] [Google Scholar]
  13. Hartz D., McPheeters D. S., Gold L. Selection of the initiator tRNA by Escherichia coli initiation factors. Genes Dev. 1989 Dec;3(12A):1899–1912. doi: 10.1101/gad.3.12a.1899. [DOI] [PubMed] [Google Scholar]
  14. Hartz D., McPheeters D. S., Traut R., Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 1988;164:419–425. doi: 10.1016/s0076-6879(88)64058-4. [DOI] [PubMed] [Google Scholar]
  15. Hausner T. P., Geigenmüller U., Nierhaus K. H. The allosteric three-site model for the ribosomal elongation cycle. New insights into the inhibition mechanisms of aminoglycosides, thiostrepton, and viomycin. J Biol Chem. 1988 Sep 15;263(26):13103–13111. [PubMed] [Google Scholar]
  16. Konan K. V., Yanofsky C. Regulation of the Escherichia coli tna operon: nascent leader peptide control at the tnaC stop codon. J Bacteriol. 1997 Mar;179(5):1774–1779. doi: 10.1128/jb.179.5.1774-1779.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kozak M. Adherence to the first-AUG rule when a second AUG codon follows closely upon the first. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2662–2666. doi: 10.1073/pnas.92.7.2662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lovett P. S., Rogers E. J. Ribosome regulation by the nascent peptide. Microbiol Rev. 1996 Jun;60(2):366–385. doi: 10.1128/mr.60.2.366-385.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luo Z., Freitag M., Sachs M. S. Translational regulation in response to changes in amino acid availability in Neurospora crassa. Mol Cell Biol. 1995 Oct;15(10):5235–5245. doi: 10.1128/mcb.15.10.5235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Luo Z., Sachs M. S. Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa. J Bacteriol. 1996 Apr;178(8):2172–2177. doi: 10.1128/jb.178.8.2172-2177.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maas W. K. The arginine repressor of Escherichia coli. Microbiol Rev. 1994 Dec;58(4):631–640. doi: 10.1128/mr.58.4.631-640.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Orbach M. J., Sachs M. S., Yanofsky C. The Neurospora crassa arg-2 locus. Structure and expression of the gene encoding the small subunit of arginine-specific carbamoyl phosphate synthetase. J Biol Chem. 1990 Jul 5;265(19):10981–10987. [PubMed] [Google Scholar]
  23. Palacián E., Vázquez D. Interaction of arginine with the ribosomal peptidyl transferase centre. Eur J Biochem. 1979 Nov;101(2):469–473. doi: 10.1111/j.1432-1033.1979.tb19741.x. [DOI] [PubMed] [Google Scholar]
  24. Pestova T. V., Hellen C. U., Shatsky I. N. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol. 1996 Dec;16(12):6859–6869. doi: 10.1128/mcb.16.12.6859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pestova T. V., Shatsky I. N., Hellen C. U. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol Cell Biol. 1996 Dec;16(12):6870–6878. doi: 10.1128/mcb.16.12.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reynolds K., Zimmer A. M., Zimmer A. Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region. J Cell Biol. 1996 Aug;134(4):827–835. doi: 10.1083/jcb.134.4.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rogers E. J., Lovett P. S. The cis-effect of a nascent peptide on its translating ribosome: influence of the cat-86 leader pentapeptide on translation termination at leader codon 6. Mol Microbiol. 1994 Apr;12(2):181–186. doi: 10.1111/j.1365-2958.1994.tb01007.x. [DOI] [PubMed] [Google Scholar]
  28. Ruan H., Shantz L. M., Pegg A. E., Morris D. R. The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element. J Biol Chem. 1996 Nov 22;271(47):29576–29582. doi: 10.1074/jbc.271.47.29576. [DOI] [PubMed] [Google Scholar]
  29. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  30. Shen W. C., Ebbole D. J. Cross-pathway and pathway-specific control of amino acid biosynthesis in Magnaporthe grisea. Fungal Genet Biol. 1997 Feb;21(1):40–49. [PubMed] [Google Scholar]
  31. Stark H., Orlova E. V., Rinke-Appel J., Jünke N., Mueller F., Rodnina M., Wintermeyer W., Brimacombe R., van Heel M. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell. 1997 Jan 10;88(1):19–28. doi: 10.1016/s0092-8674(00)81854-1. [DOI] [PubMed] [Google Scholar]
  32. Van Duyne G. D., Ghosh G., Maas W. K., Sigler P. B. Structure of the oligomerization and L-arginine binding domain of the arginine repressor of Escherichia coli. J Mol Biol. 1996 Feb 23;256(2):377–391. doi: 10.1006/jmbi.1996.0093. [DOI] [PubMed] [Google Scholar]
  33. Wang Z., Sachs M. S. Arginine-specific regulation mediated by the Neurospora crassa arg-2 upstream open reading frame in a homologous, cell-free in vitro translation system. J Biol Chem. 1997 Jan 3;272(1):255–261. doi: 10.1074/jbc.272.1.255. [DOI] [PubMed] [Google Scholar]
  34. Werner M., Feller A., Messenguy F., Piérard A. The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell. 1987 Jun 19;49(6):805–813. doi: 10.1016/0092-8674(87)90618-0. [DOI] [PubMed] [Google Scholar]
  35. Wolin S. L., Walter P. Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J. 1988 Nov;7(11):3559–3569. doi: 10.1002/j.1460-2075.1988.tb03233.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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