Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1990 Dec;9(12):4135–4143. doi: 10.1002/j.1460-2075.1990.tb07636.x

A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

B Papadopoulou 1, P Dekker 1, J Blom 1, L A Grivell 1
PMCID: PMC552187  PMID: 1701144

Abstract

Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed.

Full text

PDF
4135

Images in this article

Selected References

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

  1. Aziz N., Munro H. N. Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8478–8482. doi: 10.1073/pnas.84.23.8478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brawerman G. Determinants of messenger RNA stability. Cell. 1987 Jan 16;48(1):5–6. doi: 10.1016/0092-8674(87)90346-1. [DOI] [PubMed] [Google Scholar]
  4. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  5. Costanzo M. C., Fox T. D. Specific translational activation by nuclear gene products occurs in the 5' untranslated leader of a yeast mitochondrial mRNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2677–2681. doi: 10.1073/pnas.85.8.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dieckmann C. L., Mittelmeier T. M. Nuclearly-encoded CBP1 interacts with the 5' end of mitochondrial cytochrome b pre-mRNA. Curr Genet. 1987;12(6):391–397. doi: 10.1007/BF00434815. [DOI] [PubMed] [Google Scholar]
  7. Dorsman J. C., van Heeswijk W. C., Grivell L. A. Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast. Nucleic Acids Res. 1988 Aug 11;16(15):7287–7301. doi: 10.1093/nar/16.15.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Draper D. E. How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins. Trends Biochem Sci. 1989 Aug;14(8):335–338. doi: 10.1016/0968-0004(89)90167-9. [DOI] [PubMed] [Google Scholar]
  9. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grivell L. A. Nucleo-mitochondrial interactions in yeast mitochondrial biogenesis. Eur J Biochem. 1989 Jul 1;182(3):477–493. doi: 10.1111/j.1432-1033.1989.tb14854.x. [DOI] [PubMed] [Google Scholar]
  11. Grivell L. A., Reijnders L., Borst P. Isolation of yeast mitochondrial ribosomes highly active in protein synthesis. Biochim Biophys Acta. 1971 Sep 30;247(1):91–103. doi: 10.1016/0005-2787(71)90811-2. [DOI] [PubMed] [Google Scholar]
  12. Groot G. S., Flavell R. A., Van Ommen G. J., Grivell L. A. Yeast mitochondrial RNA does not contain poly(A) Nature. 1974 Nov 8;252(5479):167–169. doi: 10.1038/252167a0. [DOI] [PubMed] [Google Scholar]
  13. Hultmark D., Klemenz R., Gehring W. J. Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22. Cell. 1986 Feb 14;44(3):429–438. doi: 10.1016/0092-8674(86)90464-2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Li M., Tzagoloff A., Underbrink-Lyon K., Martin N. C. Identification of the paromomycin-resistance mutation in the 15 S rRNA gene of yeast mitochondria. J Biol Chem. 1982 May 25;257(10):5921–5928. [PubMed] [Google Scholar]
  18. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ooi B. G., Lukins H. B., Linnane A. W., Nagley P. Biogenesis of mitochondria: a mutation in the 5'-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex. Nucleic Acids Res. 1987 Mar 11;15(5):1965–1977. doi: 10.1093/nar/15.5.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  21. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  22. Romaniuk P. J., Lowary P., Wu H. N., Stormo G., Uhlenbeck O. C. RNA binding site of R17 coat protein. Biochemistry. 1987 Mar 24;26(6):1563–1568. doi: 10.1021/bi00380a011. [DOI] [PubMed] [Google Scholar]
  23. Rödel G., Körte A., Kaudewitz F. Mitochondrial suppression of a yeast nuclear mutation which affects the translation of the mitochondrial apocytochrome b transcript. Curr Genet. 1985;9(8):641–648. doi: 10.1007/BF00449816. [DOI] [PubMed] [Google Scholar]
  24. Schimmel P. RNA pseudoknots that interact with components of the translation apparatus. Cell. 1989 Jul 14;58(1):9–12. doi: 10.1016/0092-8674(89)90395-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thomas M. S., Nomura M. Translational regulation of the L11 ribosomal protein operon of Escherichia coli: mutations that define the target site for repression by L1. Nucleic Acids Res. 1987 Apr 10;15(7):3085–3096. doi: 10.1093/nar/15.7.3085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 1987;154:329–350. doi: 10.1016/0076-6879(87)54083-6. [DOI] [PubMed] [Google Scholar]
  28. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES