Change in RNAPII Protein Structure Guides Production of RNA from DNA

"This so-called 'polymerase pausing' occurs when RNAPII literally stops soon after beginning transcription for a short period before starting up again," explained Dr. Ott, who is also a professor of medicine at the University of California, San Francisco, with which Gladstone is affiliated. "All we knew was that this behavior was important for the precise transcription of DNA into RNA, so we set out to understand how, when and—most importantly—why."

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The research team focused their efforts on a segment of RNAPII called the C-terminal domain, or CTD. This section is most intimately involved with transcription regulation. Previous research had found that CTD's chemical structure is modified before and during transcription. However, the combinations of modifications as well as precisely how they influence or control transcription remained unclear. So in laboratory experiments on cells extracted from mammals, the researchers took a closer look.

The first breakthrough came when the research team identified a new type of modification, known as acetylation, which regulated transcription.

"Our next breakthrough occurred when we pinpointed the precise locations on the CTD where acetylation occurred—and realized it was unique to higher eukaryotes," explained Sebastian Schröder, PhD, the paper's first author. "We then wanted to see how this mammalian-specific acetylation fit into the realm of polymerase pausing."

Now that the team knew where the CTD became acetylated, their next goal was to find out when. Clues to the timing of acetylation came in experiments where they mutated RNAPII so that CDT was unable to become acetylated. In these cases, the length of polymerase pausing dropped, and the necessary steps for the completion of transcription failed to occur. Additional experiments revealed the elusive timeline of acetylation and transcription.

"RNAPII binds to DNA to prepare for transcription. Shortly after that we see polymerase pausing—at which point the CTD becomes highly acetylated," continued Dr. Shröder. "Soon after the pause, CTD is then deacetylated—the original modification is reversed—and transcription continues without a hitch."

Polymerase pausing is not unique to mammals—in fact it was characterized in HIV, the virus that causes AIDS, many years ago—but the fact that the CTD becomes acetylated just before or during the time when transcription is paused appears to be unique. Drs. Ott and Schröder argue that CTD acetylation is a stabilizer, preparing RNAPII for efficient completion of transcription and slowing down the process to make sure everything is functioning correctly—not unlike the final 'systems check' a pilot must perform before takeoff.

These findings offer important insight into the relationship between acetylation and transcription. And given the importance of transcription in the growth and maturation of cells in general, the team's result stands to inform scientists about a variety of cellular processes. These include, for example, the mechanisms behind stem-cell development and what happens when normal cellular growth spirals out of control, such as in cancer.

"However, there is still much we don't know about acetylation as it relates to transcription," said Dr. Ott. "For example, if CTD acetylation is important for stabilizing transcriptional pausing, why do we also see CTD acetylation at non-paused genes, although at different locations? Further, we believe there may be other steps in the transcription cycle that depend upon acetylation. Our most immediate goal is to find them. By doing so, we hope to deepen our understanding of one of nature's most elegant biological processes."

Source: Eurekalert