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

ex enables transcription factors and other regulatory proteins to influence the RNA polymerase II that actually performs the transcription.

To understand precisely how Mediator does its job, scientists have needed an accurate 3-D model of its architecture, including the locations of all its subunit proteins and a description of the different conformations Mediator can adopt to influence interactions between other components of the transcription machinery.

However, Mediator is enormous by biological standards: the version found in yeast has 25 distinct protein subunits and the human version has 30. It is also highly flexible. That combination of large size, high complexity and high flexibility makes it a poor candidate for high resolution imaging methods such as X-ray crystallography or nuclear magnetic resonance spectroscopy.

As a postdoctoral researcher in the Kornberg laboratory in the 1990s, Asturias helped pioneer the use of "single particle" electron microscopy (EM) for the imaging of large transcription complexes such as Mediator. Single-particle EM requires the taking of thousands of separate EM images of a particle of interesttypically very "noisy" images, which depict a particle in different orientations and perhaps also in a number of different conformations. All these data must be filtered and averaged to reduce the noise and yield useful 3-D pictures. In a 1999 study in Science, Asturias and colleagues used an early form of single-particle EM to determine the first rough structure of the full Mediator complex.

In the decade and a half since then, Asturias's group has continued to use EM techniques to study Mediator. Others have used high-resolution techniques to study individual Mediator subunits or portions of the complex. However, a clear and accurate picture of how the whole structure fits together has been elusive until now.

Turning a Model on its Head

To determine the full structure
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Contact: Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
Source:Eurekalert  

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