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Scientists generate long-sought molecular map of critical genetic machinery
Date:6/5/2014

clearly, Asturias and his colleagues began by producing highly pure quantities of a standard yeast version of Mediatorthe purification process itself being a major challenge. They then used this collection of Mediator particles to record roughly 85,000 EM images, which they categorized according to conformation. Averaging these yielded the clearest 3D model yet of the Mediator structure, to a resolution of about 18 Angstroms (1.8 billionths of a meter).

Using various other biochemical analyses, including the subtraction of different protein subunits to see how the EM images changed, the scientists were able to identify the precise locations of yeast Mediator's 25 protein subunits.

This mapping resulted in a comprehensive revision of the old rough model of Mediator's head-middle-tail structure. "After we located all the protein subunits, we realized that the head module is at the top of Mediator, not the bottom as had been thought," said Kuang-Lei Tsai, a postdoctoral fellow in the Asturias Laboratory, who was first author of the study. "These new data have helped us make sense of many previous biochemical observations."

Asturias and Tsai next collaborated with the laboratory of Joan and Ron ConawayJoan is another Kornberg alumnusat the Stowers Institute for Medical Research in Kansas City. The Conaway team had been working on human Mediator and now provided pure samples for EM imaging, as well as biochemical analyses of the subunit locations.

This work revealed that human Mediator shares the same broad architecture, implying that this structure has been, for the most part, conserved throughout the billion years of evolution that separate yeast and humans. "Basically the two Mediators have similar overall structure," said Tsai.

In the last part of the study, Asturias and Tsai used the new structural data to show how Mediator likely changes its conformation as it interacts with RNA polymerase on the one hand, and various t
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Contact: Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
Source:Eurekalert  

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