Bringing it all together, we propose a model for TFIIIC–TFIIIB assembly. On this tRNAHis gene, TFIIIC binds the A-box and B-box. τB stably interacts with the B-box, while τA dynamically binds upstream. TFIIIB induces DNA bending potentially in a similar way as in type I promoter.

To further investigate the A-box's role, we used footprinting assays to examine its impact on τA positioning and downstream effects. While the WT A-box enables TFIIIB-induced DNA bending, the A-box mutant disrupts τA positioning, highlighting its role in TFIIIB recruitment.

Using these insights, we applied the smFM system to test the roles of the A-box and B-box in TFIIIC binding. Mutating both motifs or using only the A-box abolished binding, confirming the B-box's key role. A-box mutations did not disrupt binding, as confirmed by EMSA.

This collaboration allowed us to tag the DBD (from τ95) and study TFIIIC’s dynamic interaction with DNA using smFM. Both τA and τB engage DNA within 200 ms.

This observation led us to hypothesize that the "blurry blob" represents the DBD (from τ95) interacting dynamically with DNA. To prove this, we started a collaboration with the @olivierduss.bsky.social
and the Twitter-less PhD student Anastasiia Chaban.

Despite identifying key τA domains and observing DNA curvature, there were still many avenues to explore. A closer look at the τA 2D classes revealed an intriguing "blurry blob" above the DNA. Could this be the elusive DNA-binding domain (DBD) missing from our structure?

While τB anchors TFIIIC via conserved interactions with the B-box, τA engages dynamically with the tRNA gene through the helical domain, WH6 in τ138, TPR15 in τ131 and the DNA-interacting loop in τ95. Pronounced DNA curvature suggests shape recognition.

After mapping the TFIIIC-promoter architecture, we focused on τB. The yeast τB structure shows a highly conserved interaction with the B-box compared to humans. Extra base-specific contacts outside the B-box hint additional role in transcription—and possibly genome organization??

We didn’t give up on solving the complete TFIIIC structure. In collaboration with @EustermannLab
(special thanks to Luis Hauptmann), we used cryo-EM single-particle mapping of distances and orientations to determine the TFIIIC-promoter architecture.

Although we obtained two separate maps, 2D class averages revealed that τB and τA are simultaneously engaged with the tRNA gene. Despite our efforts, a 3D map of the full TFIIIC complex eluded us. Perhaps someone else can solve it? All datasets will soon be available in EMPIAR.

In our previous study, the human TFIIIC structure showed how τB recognizes the B-box, however how τA binds the A-box was not resolved. Using the yeast system we aimed to answer this. After collecting nearly 80k micrographs, we resolved τA (3.7 Å) and τB (2.5 Å)—BOTH bound to DNA

This is my first post here :) Our TFIIIC story continues with our latest work in NAR (academic.oup.com/nar/advance-...). Using cryo-EM and single-molecule fluorescence microscopy (smFM), we explored unanswered questions in our human TFIIIC structure. Want to see the updated TFIIIC structure?