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Molecular basis of FIR-mediated c-myc transcriptional control

Abstract

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.

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Figure 1: FIR and FBP are nucleic acid–binding proteins involved in c-myc transcription regulation.
Figure 2: FIR and FBP binding to ssFUSE DNA.
Figure 3: DNA binding and protein dimerization by FIR RRM1-RRM2.
Figure 4: FBP Nbox interacts with FIR RRM2 using a sparsely packed hydrophobic surface.
Figure 5: FIR–FBP interaction represents a novel recognition mode in the RRM family.
Figure 6: FIR RRM1-RRM2 independently binds FBP Nbox and ssFUSE on two physically separated sites, located on opposite sides of the molecule.
Figure 7: FBP Nbox recruits FIR to ssFUSE DNA.

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Acknowledgements

We would like to thank A. Oregioni and T. Frenkiel for help in recording NMR experiments, C. deChiara and G. Nicastro for advice on the ARIA protocols used in structure calculations, A.M. Candel for help with spectroscopic data, S. Kralovicova for general support in the lab, P. Rosenthal for useful discussions and S. Kindler (Univ. of Hamburg) for the gift of a plasmid with the FIR RRM1-RRM2 gene. All NMR spectra were recorded at the Medical Research Council Biomedical NMR Centre. This work has been funded by the Medical Research Council Grant-in-Aid U117574558.

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Contributions

D.H. and C.D.C. performed cloning; C.D.C. and D.H. performed expression and purification of the FBP and FIR constructs; C.D.C. and G.K. recorded NMR spectra; C.D.C. analyzed NMR spectra, calculated the structures in this paper and performed all NMR titrations;. I.D.-M. performed the SIA analysis; A.R. recorded CD data; S.R.M. analyzed CD data; C.D.C. and S.R.M. recorded and analyzed BLI data; C.D.C. and A.R. wrote the paper; all authors were involved in planning the experiments.

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Correspondence to Andres Ramos.

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Supplementary Figures 1–7, Supplementary Tables 1 and 2, Supplementary Results and Supplementary Methods (PDF 4613 kb)

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Cukier, C., Hollingworth, D., Martin, S. et al. Molecular basis of FIR-mediated c-myc transcriptional control. Nat Struct Mol Biol 17, 1058–1064 (2010). https://doi.org/10.1038/nsmb.1883

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