Stanford scientist learns how to manipulate proofreading mechanism of polymerase


CONTEXT: Using laser light to "trap" a single molecule and study its movement under a microscope, investigators at Stanford University have produced the strongest evidence yet that RNA polymerase (RNAP), the enzyme that copies genes from DNA onto RNA, has a proofreading mechanism that permits error correction.

RESULTS: In a series of studies involving single RNAP molecules from Escherichia coli, scientists found that RNAP moves along DNA in fits and starts, pausing up to 1000 times in the course of copying DNA to RNA. The majority of the pauses last between one and five seconds, but about 3% last from 20 seconds to 30 minutes.

The long pauses occur only during backtracking, the hypothetical proofreading mechanism that allows RNAP to correct errors in the copying process. The reason for the short pauses remains unknown. Findings were published in Dec. 11, 2003 issue of Nature.

IMAGE: Joshua Shaevitz, a physics graduate student at Stanford, and colleagues used 2D optical force clamps to create a laser trap that allowed them to follow a single molecule of RNAP as it traversed a DNA molecule. Two beads (blue) were held in separate optical traps (red). The smaller bead (right) was bound to a simple molecule of RNA, while the larger bead was bound to the downstream end of the DNA by noncovalent linkages (yellow). During transcriptional elongation, the beads can be seen pulled together without magnification. (Provided by J. Shaevitz, reprinted with permission from Nature)

IMPLICATIONS: The few base-pair backtracking measurements made by the group were the smallest ever made on single molecules, according to Shaevitz. The technique pushes the resolution limit for visualizing single-protein motors as they move along a single substrate.

"Our main conclusion is that RNA polymerase is possibly able to correct mistakes it makes in the RNA it is producing," Shaevitz said. "If something goes wrong in the copying process, then the wrong genetic message is created, and that leads to production of the wrong proteins and diseased cells."

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