ScienceDaily (Nov. 28, 2009) — Scientists who study RNA have faced a formidable roadblock: trying to examine RNA's movements in a living cell when they can't see the RNA. Now, a new technology has given scientists the first look ever at RNA in a live bacteria cell -- a sight that could offer new information about how the molecule moves and works.
These are fluorescent images of E. coli bacterial cells with visualized RNA. The bar denotes 2 microns. (Credit: Image courtesy of Natalia E. Broude, Ph.D. / Department of Biomedical Engineering, Boston University)
Interest in RNA, which plays a key role in manufacturing proteins, has increased in recent years, due in large part to its potential in new drug therapies. RNA localization and movement in bacterial cell are poorly understood. The problem has been finding a way to mark RNA in a living cell so that scientists can track it, says Natasha Broude, a research associate professor at Boston University's Department of Biomedical Engineering.
"You can label any protein within the cell and watch what it is doing," says Broude, a senior researcher on the new study, published in a recent issue of the Proceedings of the National Academy of Sciences. "For RNA it was much more difficult because RNA is more mobile and less stable than both proteins and DNA."
Before now, scientists used green fluorescent protein (GFP) to label RNA in a cell. But proteins were also tagged with GFP and their fluorescence was so bright, it drowned out the glow from the RNA. "The initial idea was to do something to allow us to decrease background fluorescence," Broude says.
In 2007, Broude and her colleagues developed a system to persuade a cell to synthesize protein in two fragments rather than a whole, which made the protein inactive. They then modified an RNA molecule, adding a small tail of RNA sequence that works like a handle, grabbing the fragments and pulling them together, which makes the protein active -- and glow bright green. The scientists can then follow the RNA as it moves through the cell.
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Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells
Maria Valencia-Burton a, Ankita Shah a, Jason Sutin a, Azra Borogovac a, Ron M. McCullough b, Charles R. Cantor a,b, Amit Meller a and Natalia E. Broude a,1
+ Author Affiliations
aCenter for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, 36 Cummington Street, Boston, MA 02215; and
bSequenom, Inc., 3595 John Hopkins Court, San Diego, CA 92121
Contributed by Charles R. Cantor, July 17, 2009 (received for review May 5, 2009)
Abstract
Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.
fluorescent protein live E. coli cells protein complementation RNA visualization
Footnotes
1To whom correspondence should be addressed. E-mail: nebroude@bu.edu
Author contributions: C.R.C., A.M., and N.E.B. designed research; M.V.-B., A.S., J.S., A.B., and R.M.M. performed research; M.V.-B., A.S., A.M., and N.E.B. analyzed data; and A.M. and N.E.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0907495106/DCSupplemental.
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