Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Non-optimal codon usage affects expression, structure and function of clock protein FRQ


Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1,2,3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4,5,6. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Codon optimization of frq results in high FRQ expression levels and loss of circadian rhythmicities.
Figure 2: FRQ activities in circadian feedback loops are impaired in the frq codon-optimized strains.
Figure 3: FRQ protein in the codon-optimized strains is less stable and more sensitive to trypsin digestion.
Figure 4: Codon optimization of the middle region of FRQ impairs FRQ phosphorylation and stabilizes FRQ.

Similar content being viewed by others


  1. Ikemura, T. Codon usage and tRNA content in unicellular and multicellular organisms. Mol. Biol. Evol. 2, 13–34 (1985)

    CAS  PubMed  Google Scholar 

  2. Plotkin, J. B. & Kudla, G. Synonymous but not the same: the causes and consequences of codon bias. Nature Rev. Genet. 12, 32–42 (2011)

    Article  CAS  Google Scholar 

  3. Drummond, D. A. & Wilke, C. O. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell 134, 341–352 (2008)

    Article  CAS  Google Scholar 

  4. Tuller, T. et al. An evolutionarily conserved mechanism for controlling the efficiency of protein translation. Cell 141, 344–354 (2010)

    Article  CAS  Google Scholar 

  5. Gu, W., Zhou, T. & Wilke, C. O. A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes. PLoS Comput. Biol. 6, e1000664 (2010)

    Article  ADS  Google Scholar 

  6. Cannarozzi, G. et al. A role for codon order in translation dynamics. Cell 141, 355–367 (2010)

    Article  Google Scholar 

  7. Heintzen, C. & Liu, Y. The Neurospora crassa circadian clock. Adv. Genet. 58, 25–66 (2007)

    Article  CAS  Google Scholar 

  8. Baker, C. L., Loros, J. J. & Dunlap, J. C. The circadian clock of Neurospora crassa. FEMS Microbiol. Rev. 36, 95–110 (2012)

    Article  CAS  Google Scholar 

  9. Cheng, P., He, Q., He, Q., Wang, L. & Liu, Y. Regulation of the Neurospora circadian clock by an RNA helicase. Genes Dev. 19, 234–241 (2005)

    Article  CAS  Google Scholar 

  10. Cheng, P., Yang, Y. & Liu, Y. Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock. Proc. Natl Acad. Sci. USA 98, 7408–7413 (2001)

    Article  CAS  ADS  Google Scholar 

  11. He, Q. et al. CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes Dev. 20, 2552–2565 (2006)

    Article  CAS  Google Scholar 

  12. Schafmeier, T. et al. Transcriptional feedback of Neurospora circadian clock gene by phosphorylation-dependent inactivation of its transcription factor. Cell 122, 235–246 (2005)

    Article  CAS  Google Scholar 

  13. Huang, G., Wang, L. & Liu, Y. Molecular mechanism of suppression of circadian rhythms by a critical stimulus. EMBO J. 25, 5349–5357 (2006)

    Article  CAS  Google Scholar 

  14. Lee, K., Loros, J. J. & Dunlap, J. C. Interconnected feedback loops in the Neurospora circadian system. Science 289, 107–110 (2000)

    Article  CAS  ADS  Google Scholar 

  15. Morgan, L. W., Greene, A. V. & Bell-Pedersen, D. Circadian and light-induced expression of luciferase in Neurospora crassa. Fungal Genet. Biol. 38, 327–332 (2003)

    Article  CAS  Google Scholar 

  16. Gooch, V. D. et al. Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock. Eukaryot. Cell 7, 28–37 (2008)

    Article  CAS  ADS  Google Scholar 

  17. Spencer, P. S., Siller, E., Anderson, J. F. & Barral, J. M. Silent substitutions predictably alter translation elongation rates and protein folding efficiencies. J. Mol. Biol. 422, 328–335 (2012)

    Article  CAS  Google Scholar 

  18. Bennetzen, J. L. & Hall, B. D. Codon selection in yeast. J. Biol. Chem. 257, 3026–3031 (1982)

    CAS  PubMed  Google Scholar 

  19. Colot, H. V., Loros, J. J. & Dunlap, J. C. Temperature-modulated alternative splicing and promoter use in the circadian clock gene frequency. Mol. Biol. Cell 16, 5563–5571 (2005)

    Article  CAS  Google Scholar 

  20. Diernfellner, A. et al. Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms. FEBS Lett. 581, 5759–5764 (2007)

    Article  CAS  Google Scholar 

  21. Liu, Y., Merrow, M. M., Loros, J. J. & Dunlap, J. C. How temperature changes reset a circadian oscillator. Science 281, 825–829 (1998)

    Article  CAS  ADS  Google Scholar 

  22. Liu, Y., Garceau, N., Loros, J. J. & Dunlap, J. C. Thermally regulated translational control mediates an aspect of temperature compensation in the Neurospora circadian clock. Cell 89, 477–486 (1997)

    Article  CAS  Google Scholar 

  23. Tang, C. T. et al. Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events. Proc. Natl Acad. Sci. USA 106, 10722–10727 (2009)

    Article  CAS  ADS  Google Scholar 

  24. Baker, C. L., Kettenbach, A. N., Loros, J. J., Gerber, S. A. & Dunlap, J. C. Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock. Mol. Cell 34, 354–363 (2009)

    Article  CAS  Google Scholar 

  25. Xu, Y., Ma, P., Shah, P., Rokas, A., Liu, Y. & Johnson, C. H. Non-optimal codon usage is a mechanism to achieve circadian clock conditionality. Nature (this issue)

  26. Zhou, T., Weems, M. & Wilke, C. O. Translationally optimal codons associate with structurally sensitive sites in proteins. Mol. Biol. Evol. 26, 1571–1580 (2009)

    Article  CAS  Google Scholar 

  27. Siller, E., DeZwaan, D. C., Anderson, J. F., Freeman, B. C. & Barral, J. M. Slowing bacterial translation speed enhances eukaryotic protein folding efficiency. J. Mol. Biol. 396, 1310–1318 (2010)

    Article  CAS  Google Scholar 

  28. Komar, A. A., Lesnik, T. & Reiss, C. Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation. FEBS Lett. 462, 387–391 (1999)

    Article  CAS  Google Scholar 

  29. Kimchi-Sarfaty, C. et al. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525–528 (2007)

    Article  CAS  ADS  Google Scholar 

  30. Johnson, C. H., Stewart, P. L. & Egli, M. The cyanobacterial circadian system: from biophysics to bioevolution. Annu. Rev. Biophys. 40, 143–167 (2011)

    Article  CAS  Google Scholar 

  31. Cha, J., Yuan, H. & Liu, Y. Regulation of the activity and cellular localization of the circadian clock protein FRQ. J. Biol. Chem. 286, 11469–11478 (2011)

    Article  CAS  Google Scholar 

  32. Aronson, B. D., Johnson, K. A., Loros, J. J. & Dunlap, J. C. Negative feedback defining a circadian clock: autoregulation in the clock gene frequency. Science 263, 1578–1584 (1994)

    Article  CAS  ADS  Google Scholar 

  33. Wright, F. The ‘effective number of codons’ used in a gene. Gene 87, 23–29 (1990)

    Article  CAS  Google Scholar 

  34. Ishihama, Y. et al. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics 4, 1265–1272 (2005)

    Article  CAS  Google Scholar 

  35. Bell-Pedersen, D., Dunlap, J. C. & Loros, J. J. Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16, 513–521 (1996)

    Article  CAS  Google Scholar 

  36. Cheng, P., Yang, Y., Heintzen, C. & Liu, Y. Coiled-coil domain mediated FRQ–FRQ interaction is essential for its circadian clock function in Neurospora. EMBO J. 20, 101–108 (2001)

    Article  CAS  Google Scholar 

  37. Garceau, N. Y., Liu, Y., Loros, J. J. & Dunlap, J. C. Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89, 469–476 (1997)

    Article  CAS  Google Scholar 

  38. Guo, J., Cheng, P., Yuan, H. & Liu, Y. The exosome regulates circadian gene expression in a posttranscriptional negative feedback loop. Cell 138, 1236–1246 (2009)

    Article  CAS  Google Scholar 

  39. Crosthwaite, S. K., Loros, J. J. & Dunlap, J. C. Light-Induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript. Cell 81, 1003–1012 (1995)

    Article  CAS  Google Scholar 

  40. Choudhary, S. et al. A double-stranded-RNA response program important for RNA interference efficiency. Mol. Cell. Biol. 27, 3995–4005 (2007)

    Article  CAS  Google Scholar 

Download references


We thank H. Yuan and Q. Ye for technical assistance, J. Dunlap for providing the pfrq-luc-I construct and M. Rosbash for suggesting the temperature experiments. We apologize to those colleagues whose studies we could not cite owing to space limitations. This work was supported by grants from the National Institutes of Health to Y.L. (GM068496 & GM062591) and M.S.S. (GM47498), and from the Welch Foundation (I-1560) to Y.L.

Author information

Authors and Affiliations



Y.L., M.Z. and J.G. designed the research. M.Z., J.G., J.C., M.C., S.C. and J.M.B. performed experiments. M.Z., J.G., J.M.B., M.S.S. and Y.L. analysed the results. Y.L. and M.Z. wrote the paper; J.G., J.M.B. and M.S.S. edited and commented on the manuscript.

Corresponding author

Correspondence to Yi Liu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 and Supplementary Tables 1-3. (PDF 2074 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhou, M., Guo, J., Cha, J. et al. Non-optimal codon usage affects expression, structure and function of clock protein FRQ. Nature 495, 111–115 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing