Life’s instructions are written in DNA, but it is the enzyme RNA polymerase II (pol II) that reads the script, transcribing RNA in eukaryotic cells and eventually giving rise to proteins. Scientists know that Pol II must advance down the gene in perfect sync with other biological processes; aberrations in the movement of this enzyme have been linked to cancer and aging. But technical hurdles prevented them from precisely determining how this important molecular machine moves along DNA, and what governs its pauses and accelerations.
A new study fills in many of those knowledge gaps. In a paper published in *Nature Structural & Molecular Biology*, researchers used a single-molecule platform to watch individual mammalian transcription complexes in action. The result is a clear view of how this molecular engine accelerates, pauses, and shifts gears as it transcribes genetic details.
> “What’s really striking is how this machine functions almost like a finely tuned automobile. It has the equivalent of multiple gears, or speed modes, each controlled by the binding of different regulatory proteins. We figured out, for the first time, how each gear is controlled.”
> Shixin liu, head of the Laboratory of Nanoscale Biophysics and Biochemistry
“We’re finally seeing where Pol II is, in both time and space, during the process,” adds Joel E. Cohen, head of the laboratory of Populations. “Our platform allowed us to objectively assess when this machine shifts gears, and how fast it goes.”
New tool provides new answers
First discovered more then fifty years ago by Rockefeller’s Robert Roeder, Pol II moves step-by-step along the DNA molecule, constructing a matching RNA strand that will ultimately give rise to proteins. But Pol II does not travel along DNA at a steady clip, especially in higher organisms like humans.
After initiation, it slows and frequently enough pauses near the start of a gene before regulatory proteins such as P-tefb and PAF1C propel it into rapid transcription mode. as it nears the end of a gene, the enzyme decelerates again to finish cleanly. this pacing is crucial: too fast or too slow, and RNA molecules cannot be properly processed or coordinated with other vital cellular events. Missteps in Pol II’s speed control have been linked to aging and a myriad of diseases including cancer.
“A lot of people aren’t aware of these links. In fact, I’m often asked: as long as it can make RNA and we know how it does it, do we really care about the speed of the machine, or whether it pauses?” Liu says. “we care precisely because we know that the kinetics of transcription are important for proper gene expression and are linked to various diseases.”
Technical limitations explain much of why prior studies struggled to unambiguously illuminate how Pol II regulates its motion. Techniques that measured averages across many molecules blurred the contributions of individual proteins, while single-molecule studies in simpler organisms such as yeast did not fully represent the intricate regulatory mechanisms of mammalian cells. Liu, an expert in single-molecule methods, realized that he could overcome these barriers only by rebuilding a mammalian transcription system *in vitro*, piece by piece from purified proteins, and pairing it with advanced imaging techniques and comp