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Scientists at St. Jude Children's Research Hospital have reconciled two closely related but contentious mechanisms underlying transcription, the process of converting genetic information in DNA into messenger RNA. Phase separation has been proposed as a driving force in transcription due to its ability to selectively concentrate proteins and DNA in discrete droplets.

However, scientists have been unclear about what really matters for transcription: the phase-separated droplets or the molecular interactions that contribute to phase separation by forming networks.

To address this question, researchers used the yeast transcription factor Gcn4 to compare the role of small, soluble protein complexes against that of larger, phase-separated droplets. today in Molecular Cell, they found that the two types of assemblies are deeply intertwined, with shared driving forces behind the formation of both. They also revealed that while transcription can occur via both mechanisms, phase separation does not necessarily offer increased activity. In fact, it can dampen it.

The entangled nature of the two models spurred collaboration between co-corresponding authors Tanja Mittag, Ph.D., St. Jude Department of Structural Biology, and Aseem Ansari, Ph.D., St. Jude Department of Chemical Biology & Therapeutics.

"We wanted to identify what is actually functional in gene regulation versus what is simply a consequence of the inherent stickiness of the sequences that tend to form networked structures," Ansari said. "We got to grapple with things that were inconvenient. And we found that it wasn't 'this or that.' It was 'this and that.'"

"While there is a lot of debate in the literature regarding which model is correct, we noticed that these two models haven't been compared head-to-head, which is exactly what we did in this study," Mittag added.

Intertwined properties give nuance and layers of control

To see if condensates were the driving force or if soluble complexes sufficed for transcription activity, the researchers methodically tweaked the ability of Gcn4 to phase separate. Since transcription involves many proteins, the researchers also examined the effect of adjusting the interaction between Gcn4 and its transcriptional binding partner, Med15. They found that increasing the Gcn4's ability to form droplets by itself did not necessarily increase activity.

Interestingly, when adding Med15 and DNA to the mix, the formation of droplets could explain the activity well, but so did the molecular interactions. Only when the strength of the interaction between Med15 and Gcn4 was very high did the droplet model clearly win.

"In general, we think condensates and complexes act very similarly," said Mittag. "However, if we generate condensates that rely on very high affinities, their internal material properties are likely not well suited for promoting biochemical activity."

Additionally, the researchers showed that DNA broke up Gcn4 condensates, contrary to the perceived supportive role of DNA in transcriptional condensate formation.

"The assumption was that if you have a piece of DNA with multiple binding sites next to each other, it enables nucleation and condensate formation," Ansari explained. "We found exactly the opposite: you could have transcription factors forming a condensate entirely on their own, and when you give them DNA, they fall apart."

The researchers showed that this was due to DNA binding to Gcn4 and driving the formation of the small, soluble complexes, thus "dissolving" the condensate.

Collectively, the findings offer a cautionary message for interpreting transcription exclusively through the lens of either model, especially considering the complex environment in which transcription occurs.

"The answers were initially not as clear cut as we had expected, but that tempered our thinking, leading us to consider that maybe these are intertwined properties, giving nuance and layers of control that the field has largely ignored," Ansari said. "It's a lesson that these mechanisms are not mutually exclusive."

The study's first author is Anne Bremer, St. Jude. The study's other authors are Walter Lang, Ryan Kempen, Kumari Sweta, Aaron Taylor and Madeleine Borgia, St. Jude.