Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Nov;11(11):5681–5692. doi: 10.1128/mcb.11.11.5681

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

M Moritz 1, B A Pulaski 1, J L Woolford Jr 1
PMCID: PMC361939  PMID: 1922070

Abstract

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.

Full text

PDF
5681

Images in this article

5688Image
on p.5688
5688Image
on p.5688

Selected References

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

  1. Andrew C., Hopper A. K., Hall B. D. A yeast mutant defective in the processing of 27S r-RNA precursor. Mol Gen Genet. 1976 Feb 27;144(1):29–37. doi: 10.1007/BF00277300. [DOI] [PubMed] [Google Scholar]
  2. Auer J., Spicker G., Böck A. Organization and structure of the Methanococcus transcriptional unit homologous to the Escherichia coli "spectinomycin operon". Implications for the evolutionary relationship of 70 S and 80 S ribosomes. J Mol Biol. 1989 Sep 5;209(1):21–36. doi: 10.1016/0022-2836(89)90167-8. [DOI] [PubMed] [Google Scholar]
  3. Baim S. B., Pietras D. F., Eustice D. C., Sherman F. A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c. Mol Cell Biol. 1985 Aug;5(8):1839–1846. doi: 10.1128/mcb.5.8.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bayliss F. T., Ingrahm J. L. Mutation in Saccharomyces cerevisiae conferring streptomycin and cold sensitivity by affecting ribosome formation and function. J Bacteriol. 1974 May;118(2):319–328. doi: 10.1128/jb.118.2.319-328.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  6. Bryant R. E., Sypherd P. S. Genetic analysis of cold-sensitive ribosome maturation mutants of Escherichia coli. J Bacteriol. 1974 Mar;117(3):1082–1092. doi: 10.1128/jb.117.3.1082-1092.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter C. J., Cannon M., Jiménez A. A trichodermin-resistant mutant of Saccharomyces cerevisiae with an abnormal distribution of native ribosomal subunits. Eur J Biochem. 1980;107(1):173–183. doi: 10.1111/j.1432-1033.1980.tb04638.x. [DOI] [PubMed] [Google Scholar]
  8. Cerretti D. P., Dean D., Davis G. R., Bedwell D. M., Nomura M. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene. Nucleic Acids Res. 1983 May 11;11(9):2599–2616. doi: 10.1093/nar/11.9.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cigan A. M., Donahue T. F. Sequence and structural features associated with translational initiator regions in yeast--a review. Gene. 1987;59(1):1–18. doi: 10.1016/0378-1119(87)90261-7. [DOI] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donahue T. F., Cigan A. M. Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region. Mol Cell Biol. 1988 Jul;8(7):2955–2963. doi: 10.1128/mcb.8.7.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fabian G. R., Hopper A. K. RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits. J Bacteriol. 1987 Apr;169(4):1571–1578. doi: 10.1128/jb.169.4.1571-1578.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg B., McLaughlin C. S., Moldave K. Analysis of temperature-sensitive mutant ts 187 of Saccharomyces cerevisiae altered in a component required for the initiation of protein synthesis. J Biol Chem. 1982 Sep 25;257(18):10846–10851. [PubMed] [Google Scholar]
  14. Fleming G., Belhumeur P., Skup D., Fried H. M. Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes. Proc Natl Acad Sci U S A. 1989 Jan;86(1):217–221. doi: 10.1073/pnas.86.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Futcher B., Carbon J. Toxic effects of excess cloned centromeres. Mol Cell Biol. 1986 Jun;6(6):2213–2222. doi: 10.1128/mcb.6.6.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gritz L. R., Mitlin J. A., Cannon M., Littlewood B., Carter C. J., Davies J. E. Ribosome structure, maturation of ribosomal RNA and drug sensitivity in temperature-sensitive mutants of Saccharomyces cerevisiae. Mol Gen Genet. 1982;188(3):384–391. doi: 10.1007/BF00330038. [DOI] [PubMed] [Google Scholar]
  17. Guthrie C., Nashimoto H., Nomura M. Structure and function of E. coli ribosomes. 8. Cold-sensitive mutants defective in ribosome assembly. Proc Natl Acad Sci U S A. 1969 Jun;63(2):384–391. doi: 10.1073/pnas.63.2.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanic-Joyce P. J., Singer R. A., Johnston G. C. Molecular characterization of the yeast PRT1 gene in which mutations affect translation initiation and regulation of cell proliferation. J Biol Chem. 1987 Feb 25;262(6):2845–2851. [PubMed] [Google Scholar]
  19. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Helser T. L., Baan R. A., Dahlberg A. E. Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide. Mol Cell Biol. 1981 Jan;1(1):51–57. doi: 10.1128/mcb.1.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Henkin T. M., Moon S. H., Mattheakis L. C., Nomura M. Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli. Nucleic Acids Res. 1989 Sep 25;17(18):7469–7486. doi: 10.1093/nar/17.18.7469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  24. Kief D. R., Warner J. R. Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae. Mol Cell Biol. 1981 Nov;1(11):1007–1015. doi: 10.1128/mcb.1.11.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kimura J., Kimura M. The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 from the moderate thermophile Bacillus stearothermophilus ribosome. FEBS Lett. 1987 Jan 1;210(1):85–90. doi: 10.1016/0014-5793(87)81303-0. [DOI] [PubMed] [Google Scholar]
  26. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozak M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8301–8305. doi: 10.1073/pnas.87.21.8301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Larkin J. C., Thompson J. R., Woolford J. L., Jr Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene. Mol Cell Biol. 1987 May;7(5):1764–1775. doi: 10.1128/mcb.7.5.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Leer R. J., van Raamsdonk-Duin M. M., Mager W. H., Planta R. J. The primary structure of the gene encoding yeast ribosomal protein L16. FEBS Lett. 1984 Oct 1;175(2):371–376. doi: 10.1016/0014-5793(84)80771-1. [DOI] [PubMed] [Google Scholar]
  32. Li H. D., Zagorski J., Fournier M. J. Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Mar;10(3):1145–1152. doi: 10.1128/mcb.10.3.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maicas E., Pluthero F. G., Friesen J. D. The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay. Mol Cell Biol. 1988 Jan;8(1):169–175. doi: 10.1128/mcb.8.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marvaldi J., Pichon J., Marchis-Mouren G. On the control of ribosomal protein biosynthesis in E. coli. IV. Studies on a temperature-sensitive mutant defective in the assembly of 50S subunits. Mol Gen Genet. 1979 Mar 27;171(3):317–325. doi: 10.1007/BF00267587. [DOI] [PubMed] [Google Scholar]
  35. Moritz M., Paulovich A. G., Tsay Y. F., Woolford J. L., Jr Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2261–2274. doi: 10.1083/jcb.111.6.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nam H. G., Fried H. M. Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation. Mol Cell Biol. 1986 May;6(5):1535–1544. doi: 10.1128/mcb.6.5.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nashimoto H., Miura A., Saito H., Uchida H. Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12. Mol Gen Genet. 1985;199(3):381–387. doi: 10.1007/BF00330746. [DOI] [PubMed] [Google Scholar]
  38. Nashimoto H., Nomura M. Structure and function of bacterial ribosomes. XI. Dependence of 50S ribosomal assembly on simultaneous assembly of 30S subunits. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1440–1447. doi: 10.1073/pnas.67.3.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nazar R. N., Yaguchi M., Willick G. E., Rollin C. F., Roy C. The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein. Eur J Biochem. 1979 Dec 17;102(2):573–582. doi: 10.1111/j.1432-1033.1979.tb04274.x. [DOI] [PubMed] [Google Scholar]
  40. Nomura M., Traub P., Guthrie C., Nashimoto H. The assembly of ribosomes. J Cell Physiol. 1969 Oct;74(2 Suppl):241+–241+. doi: 10.1002/jcp.1040740428. [DOI] [PubMed] [Google Scholar]
  41. Ohkubo S., Muto A., Kawauchi Y., Yamao F., Osawa S. The ribosomal protein gene cluster of Mycoplasma capricolum. Mol Gen Genet. 1987 Dec;210(2):314–322. doi: 10.1007/BF00325700. [DOI] [PubMed] [Google Scholar]
  42. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pichon J., Marvaldi J., Marchis-Mouren G. On the control of ribosomal protein biosynthesis in E. coli. III. Studies on a temperature-sensitive mutant defective in the assembly of 30S subunits. Mol Gen Genet. 1979 Mar 27;171(3):307–316. doi: 10.1007/BF00267586. [DOI] [PubMed] [Google Scholar]
  44. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  45. Rotenberg M. O., Moritz M., Woolford J. L., Jr Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes. Genes Dev. 1988 Feb;2(2):160–172. doi: 10.1101/gad.2.2.160. [DOI] [PubMed] [Google Scholar]
  46. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  47. Sachs A. B., Davis R. W. Translation initiation and ribosomal biogenesis: involvement of a putative rRNA helicase and RPL46. Science. 1990 Mar 2;247(4946):1077–1079. doi: 10.1126/science.2408148. [DOI] [PubMed] [Google Scholar]
  48. Schnier J., Nishi K. Temperature-sensitive mutants with alterations in ribosomal protein L24 and isolation of intra- and extragenic suppressor mutants. Methods Enzymol. 1988;164:706–709. doi: 10.1016/s0076-6879(88)64079-1. [DOI] [PubMed] [Google Scholar]
  49. Singh A., Manney T. R. Genetic analysis of mutations affecting growth of Saccharomyces cerevisiae at low temperature. Genetics. 1974 Aug;77(4):651–659. doi: 10.1093/genetics/77.4.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stern S., Powers T., Changchien L. M., Noller H. F. RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science. 1989 May 19;244(4906):783–790. doi: 10.1126/science.2658053. [DOI] [PubMed] [Google Scholar]
  51. Surguchov A. P., Fominykch E. S., Smirnov V. N., Ter-Avanesyan M. D., Mironova L. N., Inge-Vechtomov S. G. Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit. Biochim Biophys Acta. 1981 Jun 26;654(1):149–155. doi: 10.1016/0005-2787(81)90148-9. [DOI] [PubMed] [Google Scholar]
  52. Tai P. C., Kessler D. P., Ingraham J. Cold-sensitive mutations in Salmonella typhimurium which affect ribosome synthesis. J Bacteriol. 1969 Mar;97(3):1298–1304. doi: 10.1128/jb.97.3.1298-1304.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tollervey D., Lehtonen H., Carmo-Fonseca M., Hurt E. C. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 1991 Mar;10(3):573–583. doi: 10.1002/j.1460-2075.1991.tb07984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Toniolo D., Meiss H. K., Basilico C. A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1273–1277. doi: 10.1073/pnas.70.4.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tsay Y. F., Thompson J. R., Rotenberg M. O., Larkin J. C., Woolford J. L., Jr Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein. Genes Dev. 1988 Jun;2(6):664–676. doi: 10.1101/gad.2.6.664. [DOI] [PubMed] [Google Scholar]
  56. Udem S. A., Warner J. R. Ribosomal RNA synthesis in Saccharomyces cerevisiae. J Mol Biol. 1972 Mar 28;65(2):227–242. doi: 10.1016/0022-2836(72)90279-3. [DOI] [PubMed] [Google Scholar]
  57. Underwood M. R., Fried H. M. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J. 1990 Jan;9(1):91–99. doi: 10.1002/j.1460-2075.1990.tb08084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Ursic D., Davies J. A cold-sensitive mutant of Saccharomyces cerevisiae defective in ribosome processing. Mol Gen Genet. 1979 Oct 1;175(3):313–323. doi: 10.1007/BF00397231. [DOI] [PubMed] [Google Scholar]
  59. Warner J. R., Mitra G., Schwindinger W. F., Studeny M., Fried H. M. Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover. Mol Cell Biol. 1985 Jun;5(6):1512–1521. doi: 10.1128/mcb.5.6.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Warner J. R. Synthesis of ribosomes in Saccharomyces cerevisiae. Microbiol Rev. 1989 Jun;53(2):256–271. doi: 10.1128/mr.53.2.256-271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Warner J. R., Udem S. A. Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae. J Mol Biol. 1972 Mar 28;65(2):243–257. doi: 10.1016/0022-2836(72)90280-x. [DOI] [PubMed] [Google Scholar]
  62. Wittekind M., Kolb J. M., Dodd J., Yamagishi M., Mémet S., Buhler J. M., Nomura M. Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis. Mol Cell Biol. 1990 May;10(5):2049–2059. doi: 10.1128/mcb.10.5.2049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Woolford J. L., Jr, Hereford L. M., Rosbash M. Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae. Cell. 1979 Dec;18(4):1247–1259. doi: 10.1016/0092-8674(79)90236-8. [DOI] [PubMed] [Google Scholar]
  64. Zitomer R. S., Walthall D. A., Rymond B. C., Hollenberg C. P. Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons. Mol Cell Biol. 1984 Jul;4(7):1191–1197. doi: 10.1128/mcb.4.7.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. elBaradi T. T., van der Sande C. A., Mager W. H., Raué H. A., Planta R. J. The cellular level of yeast ribosomal protein L25 is controlled principally by rapid degradation of excess protein. Curr Genet. 1986;10(10):733–739. doi: 10.1007/BF00405095. [DOI] [PubMed] [Google Scholar]
  66. van der Zeijst B. A., Kool A. J., Bloemers H. P. Isolation of active ribosomal subunits from yeast. Eur J Biochem. 1972 Oct 17;30(1):15–25. doi: 10.1111/j.1432-1033.1972.tb02066.x. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES