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

LA JOLLA, CAJune 5, 2014A team led by researchers at The Scripps Research Institute (TSRI) has used advanced electron microscopy techniques to determine the first accurate structural map of Mediator, one of the largest and most complex "molecular machines" in cells.

Mediator is crucial for the regulation of most genes' activity and works in the cells of all plants and animals. The mapping of its structurewhich includes more than two dozen unique protein subunitsrepresents a significant advance in basic cell biology and should shed light on medical conditions involving Mediator's dysfunction, from cancer to inherited developmental disorders.

The finding demonstrates how recently developed molecular imaging methods can be applied to characterize large and important protein complexes.

"Being able to determine how these large molecular machines look, how they're organized and how they move, will be critical for a better understanding of many key processes in cells," said TSRI Associate Professor Francisco J. Asturias, the senior author of the study, which was published on June 5, 2014 by the journal Cell.

A Complex Machine

The detailed map of Mediator comes nearly 20 years after the complex was first described by Stanford University biologist Roger Kornberg and colleagues. Kornberg, whose lab members at the time included Asturias, later won a Nobel Prize for his work on the gene transcription machinery of cells.

This gene transcription machinery evolved to perform one of the most basic and routine functions in biology, namely the copying of the information encoded in the DNA of genes into portable RNA "transcripts"some of which stay and work in the cell nucleus, while others exit the nucleus and are translated into proteins.

Each cell has its own pattern of gene transcription activity, determined by a regulatory system in which Mediator plays an indispensable role. The huge Mediator complex 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 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 transcription regulators on the other.

"This study has given us a fairly definitive picture of the Mediator architecture and how the different subunits are organized, so we can start to work towards an atomic resolution model," Asturias said. "We also want to understand better how Mediator interacts with all those other proteins to actually carry out transcription in a regulated manner."


'/>"/>

Contact: Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
Source:Eurekalert  

Related medicine news :

1. Scripps Florida scientists unravel the molecular secret of short, intense workouts
2. New nanomedicine by NTU and SERI scientists to bring relief to glaucoma patients
3. Scripps Research Institute scientists win $13 million grant in AIDS vaccine effort
4. Mice with mohawks help scientists link autism to 2 biological pathways in brain
5. Temple scientists receive $11.5 million grant for heart failure research
6. TSRI scientists catch misguided DNA-repair proteins in the act
7. Scientists seek answers with space station thyroid cancer study
8. MIPT scientists develop algorithm for anti-aging remedy search
9. Scientists reveal structural secrets of enzyme used to make popular anti-cholesterol drug
10. Scientists slow brain tumor growth in mice
11. UBC scientists find new way to mobilize immune system against viruses
Post Your Comments:
*Name:
*Comment:
*Email:
Related Image:
Scientists generate long-sought molecular map of critical genetic machinery
(Date:2/10/2016)... ... February 10, 2016 , ... Ongoing news of the ravages ... Association™ (ALCA) to conduct a survey that takes a closer look at cases of ... the prevalence and causes of TBI among the aging population, and identifies the challenges ...
(Date:2/10/2016)... ... February 10, 2016 , ... United Benefit Advisors (UBA), the ... addition to its growing list of Partner Firms. S.S. Nesbitt is headquartered ... to Huntsville and in between. , Harnessing the experience and insights of the ...
(Date:2/10/2016)... , ... February 10, 2016 , ... AxoGen, Inc. (NASDAQ: ... report results for the fourth quarter and full year ended December 31, 2015 on ... conference call and webcast for the investment community following the release at 4:30 PM ...
(Date:2/10/2016)... ... ... AHRA: The Association for Medical Imaging Management announced today ... as keynote speaker at the organization’s 2016 Spring Conference. Fox’s topic, Lead with ... with their own organizational staff and leadership. , “I am so excited ...
(Date:2/10/2016)... ... February 10, 2016 , ... For additional information contact ... , Pioneering book "Better with Age: The Ultimate Guide to Brain Training" by award-winning ... improve memory. The book’s publication date is March 16, 2016. A free review ...
Breaking Medicine News(10 mins):
(Date:2/10/2016)... , February 10, 2016 --> --> ... Japan .  --> Japan .  --> A ... With submission, Shire continues to strengthen ... With submission, Shire continues to strengthen its presence ... submission, Shire continues to strengthen its presence ...
(Date:2/10/2016)... SEATTLE , Feb. 10, 2016  Resolve ... potentially transformative new approaches to the treatment of ... completion of a multiple ascending dose study in ... lead compound RSLV-132. --> ... double-blind, placebo-controlled multiple ascending dose study of RSLV-132 ...
(Date:2/10/2016)... YORK , Feb. 10, 2016 Immune ... biopharmaceutical company, announced today that it has filed a ... Leukemia and other cancers. --> ... treating cancer by administration of Ceplene (histamine dihydrochloride) in ... provides methods of predicting the efficacy of Ceplene and ...
Breaking Medicine Technology: