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TRAPping proteins that work together inside living cells
Date:6/15/2009

ers of a protein of interest, Mayer and her colleagues build a tiny molecule called a tag into the initial protein. They then add a small molecule called TRAP to the living cell, which finds and fits into the tag like two pieces in a puzzle. TRAP waves around, bumping into nearby proteins. The scientists control TRAP with a flash of light, causing it to stick to coworkers it bumps into. The researchers then identify the new "TRAPped" proteins in subsequent analyses.

To demonstrate how well this method works, Mayer and colleagues tested it out on RNA polymerase, a well-studied machine in cells. The polymerase is made up of many proteins that cooperate to translate DNA. One of the polymerase proteins has a tail that is known to touch the DNA and some helper proteins just before the polymerase starts translating. No one knew if this tail -- also known as the C-terminus of the alpha subunit -- touches anything else in the core of the RNA polymerase complex.

The team engineered a tag in the C-terminus and cultured bacteria with the tagged RNA polymerase. After adding TRAP to the cells and giving it time to find the C-terminus tag, the team shined a light on the cultures.

The team then identified the proteins marked with TRAP using instruments in EMSL, DOE's Environmental Molecular Sciences Laboratory on the PNNL campus. They found that the tagged protein, as expected, interacts with many other proteins, for example previously identified helper proteins, so-called transcription factors. But they also found it on another core protein called the beta subunit, suggesting the tail of the alpha subunit makes contact with the beta subunit as it plugs along. This interaction had never been seen before.

"No one knows what the polymerase looks like when it is running," said Uljana Mayer. "Here we see the C-terminus swings back to grab the beta subunit once the polymerase starts working."

The team report their results June 15 in the j
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Contact: Mary Beckman
mary.beckman@pnl.gov
509-375-3688
DOE/Pacific Northwest National Laboratory
Source:Eurekalert  

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