Humans have it. So does Drosophila. But not yeast. That “it” is a small pause at the start of gene activity – a brief molecular halt that may have helped life evolve from simple cells to complex animals.

A new study by Charles Danko, associate professor in life science and technology at Cornell’s Baker Institute for Animal Health and in the Department of Biomedical Sciences in the College of Veterinary Medicine, and colleagues explores how this key step in gene regulation – promoter-proximal pausing – evolved across species.

Promoter-proximal pausing occurs just after a cell’s molecular “copy machine”– RNA polymerase II – is activated. The polymerase temporarily stops, usually after about 20 to 60 nucleotides or “letters” of the gene, waiting for further signals.

“According to the literature, that might be for between one and 10 minutes, which is just absolutely an eternity in the life of these proteins,” Danko said.

This checkpoint in gene regulation was first described in the 1980s by John Lis, the Barbara McClintock Professor in the Department of Molecular Biology and Genetics (College of Agriculture and Life Sciences) and one of the co-authors of the paper.

“A lot of research focused on yeast at the time, and it did not show the step of promoter-proximal pausing, so many people thought that it was not a very important process,” Danko said.

But Lis’ postdoctoral trainee, Karen Adelman, now the Edward S. Harkness Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, went on to show that such pauses exist not only in flies but also in human cells.

“So that led us to the question: When did pausing evolve?” Danko said. “When was it added as a new step in the transcription cycle, and how did it happen without disrupting transcription globally? Those are the sorts of questions this paper tackles.”

Using a technique called PRO-seq, based on methods developed in Lis’ lab, the study mapped this process in organisms across the tree of life – from bacteria and single-celled eukaryotes to plants and animals – to track evolutionary patterns. 

The team found that a weak, short-lived version of pausing already existed in simple single-celled organisms. Over evolutionary time, the pause became longer and more precisely positioned in animals, thanks to the emergence of new protein complexes that stabilize and regulate it, most notably the negative elongation factor (NELF).

Danko explained that four NELF sub-units appeared at different points in evolution. Two core sub-units were present in many eukaryotes, while the other two emerged later, allowing RNA polymerase to pause for longer and giving cells more precise control over gene activity.

“I think this is a really exciting finding, because it gives a lot of context on when these particular pausing systems evolved,” Danko said.

To test the importance of NELF, the Cornell researchers collaborated with colleagues at the Memorial Sloan Kettering Cancer Center, depleting two of the complex’s subunits in mouse cells. Without these regulators, RNA polymerase moved too far along genes too quickly, and many genes failed to respond properly to heat stress, which was meant to induce transcription of a core set of heat shock genes via the transcription factor HSF.

“We found that in bulk, a lot of genes don’t get up-regulated to the same extent as they did when NELF was intact,” Danko said.

Danko compared NELF’s role to adding knobs on a stereo, allowing cells to fine-tune the “volume” of gene expression.

“This ties back to the important step in the development of multicellular animal – being able to control gene activity exquisitely well,” Danko said. “We think the pause does this, and the evolution of NELF proteins in common ancestors leading up to the rise of multicellular animals supports this notion.”

These small moments can have far-reaching consequences. By adjusting when and how genes are turned on, promoter-proximal pausing helps keep cells functioning properly – a control that, when disrupted, can contribute to diseases like cancer.

“It’s really important to understand what drives transcription if we’re ever going to get a handle on these connections with disease,” Danko said. “Otherwise all we have is a list of genes that’s changed in these diseases, without really understanding their underlying significance.”

Additional Cornell faculty co-authors on the paper are: Anna-Katerina Hadjantonakis, professor at Weill Cornell Medicine; Ilana L. Brito, associate professor in the Meinig School of Biomedical Engineering at Cornell Engineering; and John Lis, Barbara McClintock Professor in the Department of Molecular Biology and Genetics at the College of Agriculture and Life Sciences. Also contributing to the paper were numerous staff, doctoral candidates and postdoctoral researchers in their labs.

The research was funded through the National Institutes of Health.

Olivia Hall is a freelance writer for the College of Veterinary Medicine.