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wolfisd.bsky.social

@wolfisd.bsky.social

40 followers 75 following 15 posts

Amateur Structural Biologist | PhD @EMBL Heidelberg Müller Group | Interested in transcription and chromatin

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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?

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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??

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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.

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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

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wolfisd.bsky.social @wolfisd.bsky.social · 11/12/2024

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?

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