Skip to main content
PMC home page
RNA logoLink to RNA
. 1996 Mar;2(3):254–263.

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

R Liu 1, S W Liebman 1
PMCID: PMC1369368  PMID: 8608449

Abstract

Recent evidence suggests that ribosomal RNAs have functional roles in translation. We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells. Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA. This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin. We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation. Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide. Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.

Full Text

The Full Text of this article is available as a PDF (5.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chernoff Y. O., Lindquist S. L., Ono B., Inge-Vechtomov S. G., Liebman S. W. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science. 1995 May 12;268(5212):880–884. doi: 10.1126/science.7754373. [DOI] [PubMed] [Google Scholar]
  2. Chernoff Y. O., Vincent A., Liebman S. W. Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics. EMBO J. 1994 Feb 15;13(4):906–913. doi: 10.1002/j.1460-2075.1994.tb06334.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Donahue T. F., Farabaugh P. J., Fink G. R. Suppressible four-base glycine and proline codons in yeast. Science. 1981 Apr 24;212(4493):455–457. doi: 10.1126/science.7010605. [DOI] [PubMed] [Google Scholar]
  4. Frolova L., Le Goff X., Rasmussen H. H., Cheperegin S., Drugeon G., Kress M., Arman I., Haenni A. L., Celis J. E., Philippe M. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature. 1994 Dec 15;372(6507):701–703. doi: 10.1038/372701a0. [DOI] [PubMed] [Google Scholar]
  5. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glück A., Endo Y., Wool I. G. The ribosomal RNA identity elements for ricin and for alpha-sarcin: mutations in the putative CG pair that closes a GAGA tetraloop. Nucleic Acids Res. 1994 Feb 11;22(3):321–324. doi: 10.1093/nar/22.3.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gould J. H., Hartley M. R., Welsh P. C., Hoshizaki D. K., Frankel A., Roberts L. M., Lord J. M. Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes. Mol Gen Genet. 1991 Nov;230(1-2):81–90. doi: 10.1007/BF00290654. [DOI] [PubMed] [Google Scholar]
  8. Grant P., Sánchez L., Jiménez A. Cryptopleurine resistance: genetic locus for a 40S ribosomal component in Saccharomyces cerevisiae. J Bacteriol. 1974 Dec;120(3):1308–1314. doi: 10.1128/jb.120.3.1308-1314.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gutell R. R., Gray M. W., Schnare M. N. A compilation of large subunit (23S and 23S-like) ribosomal RNA structures: 1993. Nucleic Acids Res. 1993 Jul 1;21(13):3055–3074. doi: 10.1093/nar/21.13.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kawakami K., Nakamura Y. Autogenous suppression of an opal mutation in the gene encoding peptide chain release factor 2. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8432–8436. doi: 10.1073/pnas.87.21.8432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Liebman S. W., Sherman F. Inhibition of growth by amber suppressors in yeast. Genetics. 1976 Feb;82(2):233–249. doi: 10.1093/genetics/82.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marchant A., Hartley M. R. Mutational studies on the alpha-sarcin loop of Escherichia coli 23S ribosomal RNA. Eur J Biochem. 1994 Nov 15;226(1):141–147. doi: 10.1111/j.1432-1033.1994.tb20035.x. [DOI] [PubMed] [Google Scholar]
  14. Melançon P., Tapprich W. E., Brakier-Gingras L. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading. J Bacteriol. 1992 Dec;174(24):7896–7901. doi: 10.1128/jb.174.24.7896-7901.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miller S. P., Bodley J. W. Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome. Nucleic Acids Res. 1991 Apr 11;19(7):1657–1660. doi: 10.1093/nar/19.7.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moazed D., Noller H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987 Jun 4;327(6121):389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
  17. Moazed D., Robertson J. M., Noller H. F. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988 Jul 28;334(6180):362–364. doi: 10.1038/334362a0. [DOI] [PubMed] [Google Scholar]
  18. Morris K. N., Wool I. G. Determination by systematic deletion of the amino acids essential for catalysis by ricin A chain. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4869–4873. doi: 10.1073/pnas.89.11.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nierhaus K. H., Schilling-Bartetzko S., Twardowski T. The two main states of the elongating ribosome and the role of the alpha-sarcin stem-loop structure of 23S RNA. Biochimie. 1992 Apr;74(4):403–410. doi: 10.1016/0300-9084(92)90118-x. [DOI] [PubMed] [Google Scholar]
  20. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  21. Noller H. F. Ribosomal RNA and translation. Annu Rev Biochem. 1991;60:191–227. doi: 10.1146/annurev.bi.60.070191.001203. [DOI] [PubMed] [Google Scholar]
  22. O'Connor M., Dahlberg A. E. Mutations at U2555, a tRNA-protected base in 23S rRNA, affect translational fidelity. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9214–9218. doi: 10.1073/pnas.90.19.9214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Poot R. A., Brink M. F., Pleij C. W., de Boer H. A., van Duin J. Separation of mutant and wild-type ribosomes based on differences in their anti Shine-Dalgarno sequence. Nucleic Acids Res. 1993 Nov 25;21(23):5398–5402. doi: 10.1093/nar/21.23.5398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rose A. B., Broach J. R. Propagation and expression of cloned genes in yeast: 2-microns circle-based vectors. Methods Enzymol. 1990;185:234–279. doi: 10.1016/0076-6879(90)85024-i. [DOI] [PubMed] [Google Scholar]
  25. Sandbaken M. G., Culbertson M. R. Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae. Genetics. 1988 Dec;120(4):923–934. doi: 10.1093/genetics/120.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Singh A., Ursic D., Davies J. Phenotypic suppression and misreading Saccharomyces cerevisiae. Nature. 1979 Jan 11;277(5692):146–148. doi: 10.1038/277146a0. [DOI] [PubMed] [Google Scholar]
  28. Stansfield I., Tuite M. F. Polypeptide chain termination in Saccharomyces cerevisiae. Curr Genet. 1994 May;25(5):385–395. doi: 10.1007/BF00351776. [DOI] [PubMed] [Google Scholar]
  29. Szewczak A. A., Moore P. B., Chang Y. L., Wool I. G. The conformation of the sarcin/ricin loop from 28S ribosomal RNA. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9581–9585. doi: 10.1073/pnas.90.20.9581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tapio S., Kurland C. G. Mutant EF-Tu increases missense error in vitro. Mol Gen Genet. 1986 Oct;205(1):186–188. doi: 10.1007/BF02428051. [DOI] [PubMed] [Google Scholar]
  31. Tapprich W. E., Dahlberg A. E. A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome. EMBO J. 1990 Aug;9(8):2649–2655. doi: 10.1002/j.1460-2075.1990.tb07447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Triman K. L. The 16S ribosomal RNA mutation database (16SMDB) Nucleic Acids Res. 1994 Sep;22(17):3563–3565. [PMC free article] [PubMed] [Google Scholar]
  33. Vijgenboom E., Vink T., Kraal B., Bosch L. Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors. EMBO J. 1985 Apr;4(4):1049–1052. doi: 10.1002/j.1460-2075.1985.tb03737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vincent A., Liebman S. W. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein. Genetics. 1992 Oct;132(2):375–386. doi: 10.1093/genetics/132.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wakem L. P., Sherman F. Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae. Genetics. 1990 Mar;124(3):515–522. doi: 10.1093/genetics/124.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weiss R. B., Murphy J. P., Gallant J. A. Genetic screen for cloned release factor genes. J Bacteriol. 1984 Apr;158(1):362–364. doi: 10.1128/jb.158.1.362-364.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wilson P. G., Culbertson M. R. SUF12 suppressor protein of yeast. A fusion protein related to the EF-1 family of elongation factors. J Mol Biol. 1988 Feb 20;199(4):559–573. doi: 10.1016/0022-2836(88)90301-4. [DOI] [PubMed] [Google Scholar]
  38. Wool I. G., Glück A., Endo Y. Ribotoxin recognition of ribosomal RNA and a proposal for the mechanism of translocation. Trends Biochem Sci. 1992 Jul;17(7):266–269. doi: 10.1016/0968-0004(92)90407-z. [DOI] [PubMed] [Google Scholar]
  39. Zhouravleva G., Frolova L., Le Goff X., Le Guellec R., Inge-Vechtomov S., Kisselev L., Philippe M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 1995 Aug 15;14(16):4065–4072. doi: 10.1002/j.1460-2075.1995.tb00078.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES