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Nanotech instruments allow first direct observations of RNA 'proofreading'

s as it pries open and copies the DNA double helix.

''Together these two papers push the study of single proteins to new limits,'' Block said. ''We've been able to achieve a resolution of three angstroms - the width of three hydrogen atoms - in our measurements of the progress of this enzyme along DNA. In so doing, we've been able to visualize a backtracking motion of just five bases that accompanies RNAP error-correction or proofreading.''

Both studies were conducted using two-dimensional optical force clamps - unique instruments designed and built by the Block lab. Located in soundproofed and temperature-controlled rooms in the basement of Stanford's Herrin labs, these devices allow researchers to trap a single molecule of RNAP in a beam of infrared light, and then watch in real time as it moves along a single molecule of DNA.

''We've been able to reduce drift and noise in our instruments to such an extent that we can see the tiniest motions of these molecules, through distances that are less than their own diameters,'' Block explained. ''Studying one macromolecule at a time, you learn so much more about its properties, but these kinds of experiments were just pipedreams 15 years ago.''

Stops and starts

In their experiments, the Block team conducted more than 300 observations of single RNAP molecules extracted from E. coli bacteria. Although structurally somewhat different from human RNAP, the E. coli enzyme plays a very similar role in the complex transfer from gene to RNA to protein.

Using the optical clamp, researchers found that RNAP does not move at a steady pace along the DNA double helix but rather undergoes a fitful series of unexplained starts and stops. ''This enzyme is either full on or full off, as far as we can tell,'' Block said. ''It moves at about ten to 15 bases a second and pauses on average about once every 100 bases. Pausing is ubiquitous. About 97 percent are short pauses that last
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Contact: Mark Shwartz
mshwartz@stanford.edu
650-723-9296
Stanford University
25-Nov-2003


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