Skip to main content
SpringerLink
Log in

Mitochondrial release factor in yeast: interplay of functional domains

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

The MRF1 gene encodes the only class I release factor found in Saccharomyces cerevisiae mitochondria, mRF1. The previously isolated point mutation mrf1-13 caused respiratory deficiency due to inhibition of mitochondrial translation. In this study, we have isolated second-site suppressors of mrf1-13. Among over 200 respiratory positive suppressor colonies, ten nuclear dominant suppressors had a new mutation in the MRF1 gene. The suppressors in combination with the original mrf1-13 revealed increased levels of mitochondrially synthesized proteins, Cox2 and Atp6. One of the suppressor alleles was cloned on a plasmid and was found to support weaker respiratory competence than in combination with mrf1-13. Finally, the possible effects of the suppressor mutations are discussed based on a structural model of mRF1 protein built for its “open” and “closed” forms using known crystal structures of prokaryotic release factor RF1 as templates. The 3D models suggest that at least some suppressors switch the structure of mRF1 from the “closed” to a permanently “open” form causing stronger binding of the mRF1 protein to the ribosome and increasing the time of ribosome occupation. This explains how the suppressor mutants may facilitate translation termination despite a defect in decoding of the stop signal.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Askarian-Amiri ME, Pel HJ, Guevremont D, McCaughan KK, Poole ES, Sumpter VG, Tate WP (2000) Functional characterization of yeast mitochondrial release factor 1. J Biol Chem 275:17241–17248

    Article  PubMed  CAS  Google Scholar 

  • Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

    Article  PubMed  CAS  Google Scholar 

  • Cormack B, Castano I (2002) Introducion of point mutations into cloned genes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Dujardin G, Pajot P, Groudinsky O, Slonimski PP (1980) Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors. Mol Gen Genet 179:469–482

    Article  PubMed  CAS  Google Scholar 

  • Fox TD (1979) Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II. Proc Natl Acad Sci USA 76:6534–6538

    Article  PubMed  CAS  Google Scholar 

  • Fox TD (1996) Genetics of mitochondrial translation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Book  Google Scholar 

  • FoxTD, Staempfli S (1982) Suppressor of the yeast mitochondrial mutations that maps in or near the 15S ribosomal RNA gene of mtDNA. Proc Natl Acad Sci USA 79:1583–1587

    Article  Google Scholar 

  • Ginalski K, Elofsson A, Fischer D, Rychlewski L (2003) 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19:1015–1018

    Article  PubMed  CAS  Google Scholar 

  • Glick BS, Pon LA (1995) Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol 260:213–223

    Article  PubMed  CAS  Google Scholar 

  • Grivell LA, Artal-Sanz M, Hakkaart G, de Jong L, Nijtmans LG, van Oosterum K, Siep M, van der Spek H (1999) Mitochondrial assembly in yeast. FEBS Lett 452:57–60

    Article  PubMed  CAS  Google Scholar 

  • Ito K, Uno M, Nakamura Y (2000) A tripeptide ‘anticodon’ deciphers stop codons in messenger RNA. Nature 403:680–684

    Article  PubMed  CAS  Google Scholar 

  • Kaczanowski R, Trzeciak L, Kucharczyk K (2001) Multitemperature single-strand conformation polymorphism. Electrophoresis 22:3539–3545

    Article  PubMed  CAS  Google Scholar 

  • Kisselev L, Ehrenberg M, Frolova L (2003) Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J 22:175–182

    Article  PubMed  CAS  Google Scholar 

  • Kjeldgaard M (2003) The unfolding story of polypeptide release factors. Mol Cell 11:8–10

    Article  PubMed  CAS  Google Scholar 

  • Klaholz BP, Pape T, Zavialov AV, Myasnikov AG, Orlova EV, Vestergaard B, Ehrenberg M, van Heel M (2003) Structure of the Escherichia coli ribosomal termination complex with release factor 2. Nature 421:90–94

    Article  PubMed  CAS  Google Scholar 

  • Maupetit J, Gautier R, Tuffery P (2006) SABBAC: online Structural Alphabet-based protein BackBone reconstruction from Alpha-Carbon trace. Nucleic Acids Res 34:W147–W151

    Article  PubMed  CAS  Google Scholar 

  • McCaughan KK, Poole ES, Pel HJ, Mansell JB, Mannering SA, Tate WP (1998) Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex. Biol Chem 379:857–866

    Article  PubMed  CAS  Google Scholar 

  • Naithani S, Saracco SA, Butler CA, Fox TD (2003) Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Mol Biol Cell 14:324–333

    Article  PubMed  CAS  Google Scholar 

  • Nakamura Y, Ito K (2002) A tripeptide discriminator for stop codon recognition. FEBS Lett 514:30–33

    Article  PubMed  CAS  Google Scholar 

  • Nissen P, Hansen J, Ban N, Moore PB, Steitz TA (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289:920–930

    Article  PubMed  CAS  Google Scholar 

  • Pei J, Sadreyev R, Grishin NV (2003) PCMA: fast and accurate multiple sequence alignment based on profile consistency. Bioinformatics 19:427–428

    Article  PubMed  CAS  Google Scholar 

  • Pel HJ, Maat C, Rep M, Grivell LA (1992) The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects. Nucleic Acids Res 20:6339–6346

    Article  PubMed  CAS  Google Scholar 

  • Petry S, Brodersen DE, Murphy FVt, Dunham CM, Selmer M, Tarry MJ, Kelley AC, Ramakrishnan V (2005) Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon. Cell 123:1255–1266

    Article  PubMed  CAS  Google Scholar 

  • Rawat U, Gao H, Zavialov A, Gursky R, Ehrenberg M, Frank J (2006) Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM. J Mol Biol 357:1144–1153

    Article  PubMed  CAS  Google Scholar 

  • Rawat UB, Zavialov AV, Sengupta J, Valle M, Grassucci RA, Linde J, Vestergaard B, Ehrenberg M, Frank J (2003) A cryo-electron microscopic study of ribosome-bound termination factor RF2. Nature 421:87–90

    Article  PubMed  CAS  Google Scholar 

  • Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  PubMed  CAS  Google Scholar 

  • Sherman F, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Shin DH, Brandsen J, Jancarik J, Yokota H, Kim R, Kim SH (2004) Structural analyses of peptide release factor 1 from Thermotoga maritima reveal domain flexibility required for its interaction with the ribosome. J Mol Biol 341:227–239

    Article  PubMed  CAS  Google Scholar 

  • Teyssier E, Hirokawa G, Tretiakova A, Jameson B, Kaji A, Kaji H (2003) Temperature-sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF). Nucleic Acids Res 31:4218–4226

    Article  PubMed  CAS  Google Scholar 

  • Towpik J, Chacinska A, Ciesla M, Ginalski K, Boguta M (2004) Mutations in the yeast mrf1 gene encoding mitochondrial release factor inhibit translation on mitochondrial ribosomes. J Biol Chem 279:14096–14103

    Article  PubMed  CAS  Google Scholar 

  • Towpik J, Kutner J, Boguta M (2005) Expression of mitochondrial release factor in relation to respiratory competence in yeast. Curr Genet 48:101–108

    Article  PubMed  CAS  Google Scholar 

  • Uno M, Ito K, Nakamura Y (2002) Polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors. Proc Natl Acad Sci USA 99:1819–1824

    Article  PubMed  CAS  Google Scholar 

  • Vestergaard B, Sanyal S, Roessle M, Mora L, Buckingham RH, Kastrup JS, Gajhede M, Svergun DI, Ehrenberg M (2005) The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure. Mol Cell 20:929–938

    Article  PubMed  CAS  Google Scholar 

  • Vestergaard B, Van LB, Andersen GR, Nyborg J, Buckingham RH, Kjeldgaard M (2001) Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1. Mol Cell 8:1375–1382

    Article  PubMed  CAS  Google Scholar 

  • Williams EH, Fox TD (2003) Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae mitochondria. RNA 9:419–431

    Article  PubMed  CAS  Google Scholar 

  • Wilson DN, Guevremont D, Tate WP (2000) The ribosomal binding and peptidyl-tRNA hydrolysis functions of Escherichia coli release factor 2 are linked through residue 246. RNA 6:1704–1713

    Article  PubMed  CAS  Google Scholar 

  • Yaffe MP, Schatz G (1984) Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci USA 81:4819–4823

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Polish Mitochondrial Network, Mitonet. K.G. was supported by EMBO Installation, NIH (1R01GM081680-01) and FNP (FOCUS) grants.

Author information

Authors and Affiliations

  1. Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106, Warsaw, Poland

    Jan Kutner, Joanna Towpik & Magdalena Boguta

  2. Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Pawinskiego 5a, 02-106, Warsaw, Poland

    Krzysztof Ginalski

Authors
  1. Jan Kutner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joanna Towpik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Krzysztof Ginalski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Magdalena Boguta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Magdalena Boguta.

Additional information

Communicated by L. Tomaska.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kutner, J., Towpik, J., Ginalski, K. et al. Mitochondrial release factor in yeast: interplay of functional domains. Curr Genet 53, 185–192 (2008). https://doi.org/10.1007/s00294-008-0177-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-008-0177-y

Keywords

Search

Navigation