KANSAS CITY, MO—Look up "transcription"—the copying of a gene's DNA into RNA intermediaries—in any old molecular biology text book, and it all seems very simple: RNA polymerase II, the enzyme that catalyzes the reaction, assembles at the start site and starts motoring down the strand, cranking out the RNA ribbon used to construct proteins. But researchers now know that RNA polymerase II often stalls on DNA strands where it was once assumed to just barrel down.
A report from the Conaway lab at the Stowers Institute for Medical Research in the July 8, 2011, edition of the journal Cell identifies a switch that allows RNA polymerase to shift gears from neutral into drive and start transcribing. This work sheds light on a process fundamental to all plant or animal cells and suggests how transcriptional anomalies could give rise to tumors.
"There are frequent roadblocks in transcriptional elongation," said Laurie Tompkins, Ph.D., who oversees transcriptional mechanisms grants at the NIH, including one that supported this work. "The discovery that MED26 plays a crucial role in overcoming one clarifies how an essential cellular process is regulated and suggests new strategies for regulating gene expression in healthy and diseased cells."
The all-Stowers team led by first author Hidehisa Takahashi, Ph.D., a post-doctoral fellow in the Conaway lab, utilized biochemistry and a mass spectrometry method called Multidimensional Protein Identification Technology, or MudPIT, to show that MED26 acts as a go-between linking transcriptional complexes carrying out seemingly disparate jobs, namely the assembly of pol II on the DNA track versus setting it in motion.
The team then showed that Med26 was required for expression of genes at which pol II was known to pause, suggesting that Med26 recruited elongation factors to jump-start the stalled polymerase at that juncture. One elongation factor shown to interact biochemically with MED26 was ELL, purified in the 90's by co-author and Stowers Investigator Ali Shilatifard, Ph.D., when he was a postdoctoral fellow in the Conaway lab at the Oklahoma Medical Research Foundation.
This work suggests that before RNA elongation, MED26 as a component of Mediator sits huddled with resting pol II, sequestering it near the start gate. Biochemists alliteratively call this behavior "pol II promoter-proximal pausing" or as Joan Conaway puts it, "It just looks like pol II can't get out of the DNA driveway and into the street." And although the polymerase occasionally lurches forward attempting to commence elongation, full-bore elongation occurs only when MED26 lets go of start-site factors and grabs onto elongation factors like ELL, releasing pol II to transcribe RNA.
An interesting corollary is that some leukemias are caused by gene translocations that create hybrids of elongation proteins like ELL and other factors —so the new connection to Mediator reported by the Conaway lab suggests that inappropriate gene expression in tumor cells could result in part from mistargeting mediator and pol II to the wrong chromosomal locations.
Why would Mother Nature would devise ornate schemes just to copy DNA into RNA? Co-author Ron Conaway, Ph.D., Investigator and co-PI of the lab, speculates that they allow fine-tuning of gene expression and as a bonus provide medicinal chemists with numerous drug targets.
Also contributing to the work were Tari J. Parmely, Shigeo Sato, PhD., Chieri Tomomori-Sato, PhD., Charles A. S. Banks, PhD., Stephanie E. Kong, PhD, Henrietta Szutorisz, PhD., from the Conaway lab; Michael P. Washburn, Ph.D. and Laurence Florens, Ph.D.and their colleagues Selene K. Swanson and Skylar Martin-Brown from the Proteomics Center; bioinformaticist Chris Seidel, Ph.D., and Chengqi Lin and Edwin R. Smith, Ph.D., in the Shilatifard lab.
The work was funded by the Stowers Institute and the National Institutes of Health.
The Stowers Institute for Medical Research is a non-profit, basic biomedical research organization dedicated to improving human health by studying the fundamental processes of life. Jim Stowers, founder of American Century Investments, and his wife Virginia opened the Institute in 2000. Since then, the Institute has spent over a half billion dollars in pursuit of its mission.
Currently the Institute is home to nearly 500 researchers and support personnel; over 20 independent research programs; and more than a dozen technology development and core facilities. Learn more about the Institute at http://www.stowers.org. Learn more about American Century Investments at http://www.americancentury.com.