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bioRxiv · 10.1101/2024.10.19.619198

Mechanism of Interaction Between the Transactivation Domain of N-MYC and the DNA-Binding Surface of TFIIIC5

Leen, E.·Yeoh, S.·Sahak, E.·Mitchell, E.·Wildsmith, G.·Batchelor, M.·Calabrese, A. N.·Büchel, G.·Bayliss, R.
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Abstract

N-myc is a member of the myc family of transcription factors, which are powerful drivers of cellular growth and consequently, important oncoproteins. N-myc interacts with many factors and complexes to affect transcription. One such complex is the RNA Polymerase III assembly factor, TFIIIC, a six-member complex that is essential for the transcription of small, structured RNA. TFIIIC and N-myc mutually restrict each others chromatin association, and their complex contributes to quality control in mRNA transcription. We previously demonstrated that the largely intrinsically disordered transactivation domain of N-myc interacts directly with a sub-complex of TFIIIC, {tau}A. Structural studies by others show that DNA binding of {tau}A is largely mediated by TFIIIC3, which suggests that TFIIIC5 is at most a secondary binding site for DNA. Here we identified the DNA binding domain of TFIIIC5 as a key binding site for N-myc. We used an integrated approach combining NMR, HDX mass spectrometry, pull-downs and biophysical assays to elucidate the molecular basis of the interaction. Two sequences in the transactivation domain of N-myc bind to the DNA binding interface of TFIIIC5. AlphaFold modelling predicts a high-confidence binding mode for the higher affinity N-myc motif that overlaps with the predicted intramolecular binding site of the C-terminal acidic plug of TFIIIC5, removal of which enhances the binding of N-myc. The same two motifs in N-myc also interact with Aurora-A kinase, which competes with N-myc for TFIIIC binding during S-phase. This model elucidates how the N-myc:TFIIIC5 interaction competes with other interactions, providing a basis for their mutual censoring function and regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/619198v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e4a76aorg.highwire.dtl.DTLVardef@ab349corg.highwire.dtl.DTLVardef@af85dorg.highwire.dtl.DTLVardef@37dd2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Leen, E., Yeoh, S., Sahak, E., Mitchell, E., Wildsmith, G., Batchelor, M., Calabrese, A. N., Büchel, G., Bayliss, R.. 2024-10-20. Mechanism of Interaction Between the Transactivation Domain of N-MYC and the DNA-Binding Surface of TFIIIC5. https://doi.org/10.1101/2024.10.19.619198

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