Technique Unveils Gene Process
BERKELEY, Calif. -- Gene transcription is the first step in the process whereby information stored in DNA patterns is used to manufacture protein. When an RNA polymerase enzyme latches onto a DNA strand and begins to slowly reel it in, the strand's genetic information is transcribed to messenger RNA. Biochemical studies of transcription appear to indicate that the RNA polymerase enzyme pauses and sometimes stops completely. But bulk biochemical measurements cannot directly measure the transcription rates, the durations of these pauses or the forces applied by the transcribing enzyme.
An optical trap and imaging technology have helped unveil the mechanisms behind gene transcription. Image processing determines the distance between two polystyrene beads attached to either end of a DNA/RNA polymerase complex. As the RNA polymerase enzyme begins reeling in the DNA strand, transcribing its store of information, scientists are able to record the history of the transcription process, one molecule at a time. Courtesy of University of California at Berkeley.
Scientists at the University of California have found an alternative approach to quantify the mechanisms of gene transcription by using an optical trap and a video microscope. An optical trap created by an 835-nm beam holds a polystyrene bead on one end of a DNA/RNA polymerase (RNAp) complex. The researchers attach a second bead to the opposite end of the strand using a micropipette and capture an image of the structure with a CCD camera. Image processing determines the distance from bead center to center, which directly relates to the progress of the gene transcription process.
As the enzyme transcribes the DNA, it mechanically reels in the extended strand, shortening the distance between the two beads. The researchers determine the bead centroid positions to an accuracy of 7 to 10 nm, corresponding to a resolution of 21 to 30 base pairs on the gene. The centroid positions represent a history of the transcription process, one molecule at a time.
Gijs Wuite and his colleagues at Berkeley and the University of Wisconsin believe that this study, reported in the March 31 issue of Science, is particularly significant because it provides a bridge between single-molecule studies and biochemistry. According to Wuite, the highly automated instrument not only makes the experiments repeatable, but also speeds the data collection rate.
A CCD camera from Watec America of Las Vegas collects images of the beads as the enzyme begins the process, and a frame grabber from Data Translation of Marlboro, Mass., digitizes the images.
Optical technology may eventually help us understand the mechanisms of gene transcription, one of the fundamental processes of life. "The next step for the technology is to obtain better resolution and to do these experiments faster," said Wuite. The complex nature of DNA enzyme processes makes faster experiments especially advantageous to gather large sets of data. Higher spatial resolution will enable the scientists to see the movement of the RNAp at a single base pair level. "This will allow us to understand the dynamics of transcription at its finest detail," Wuite added.
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