Scientists at Berkeley have reported the first direct observations of what happens when the message of a gene is being read during the actual transcription of single DNA molecules. Using a unique experimental setup they designed themselves, the researchers followed transcription by single molecules of RNA polymerase (RNAP), the enzyme responsible for reading the genetic message in humans and other higher organisms as well as bacteria. Their observations provide new insights into how genetic expression in cells may be regulated.
Carlos Bustamante, a biophysicist who holds a joint appointment with the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley (UC Berkeley), heads the group that published the results of this work in the March 31 issue of the journal Science. In addition to Bustamante, the other authors were John Davenport and Gijs Wuite with UC Berkeley and Robert Landick with the University of Wisconsin at Madison.
"Our studies reveal that RNAP molecules possess different intrinsic transcription rates and different propensities to pause and stop," the authors state in their paper. "The conformational metastability of RNAP revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene expression in both bacteria and eukaryotes."
The genetic messages stored within the DNA inside the nucleus of a living cell are read by the RNAP enzyme and transcribed into messenger RNA (mRNA) which then carries the information out of the nucleus and into the cytoplasm where protein assembly takes place. RNAP transcribes a gene by tightly clamping itself to the DNA and elongating-growing out-over the gene's base-pairs, catalyzing the creation of an mRNA template in the process.
Previous studies have shown that this elongational growth/transcription is discontinuous; the RNAP may pause at various points along the way for a few seconds or a few minutes. Transcription may even stop entirely, with the RNAP going into a state of arrest in which it neither detaches itself from the DNA nor releases its mRNA. These previous studies however, were "bulk biochemistry experiments," performed on batches of molecules. Conclusions were based on an averaging out of observations as if the activity of each molecule in the experiment is the same-an iffy assumption given the size and complexity of an enzyme like RNAP. Furthermore, the observed arrests in these bulk studies were artificially induced by solution conditions.
"Ours is the first study in which pause dynamics of RNAP have been studied systematically at a single molecule level and on enzymes that were actively transcribing," says Bustamante. "Single molecule experiments can provide a unique look into the molecular mechanisms responsible for mechano-chemical conversion processes and reveal behavior which would be averaged out in traditional bulk experiments."
The Berkeley team made their observations using an "integrated laser trap/flow control video microscope" which was designed and built under the direction of Bustamante, who heads the Advanced Microscopies Department for Berkeley Lab's Physical Biosciences Division. In this setup, a strand of DNA is tethered between two micron-sized polystyrene beads in the middle of a chamber, one bead anchored on the tip of a glass pipette and the other held by a laser beam in an optical trap. Controlling the exchange and flow of liquids through the chamber and manipulating the laser trap can then be used to apply force on the beads, and the results can be measured through video microscopy.
This unique experimental system enabled the researchers to make direct observations of the RNAP transcription process in real time over a long length of DNA (more than a thousand base-pairs). Their spatial and temporal resolution was better than previous efforts and they could record pausing and other transcription events not only as a product of solution conditions but also as a function of applied force.
In their Science paper, the researchers report that the rate at which RNAP transcription took place did vary, but the variations were not associated with specific locations along the DNA. Rather, individual RNAP molecules would switch back and forth from transcribing at a faster or slower rate, as if, Bustamante says, "they exist in different gears corresponding to different functional or dynamic states."
It was also observed that RNAP molecules transcribing at a faster rate are less likely to pause than those transcribing at a slower rate. The authors say this suggests there is a "kinetic competition" between transcription and pausing, which would mean that pauses are not intermediate steps in the transcriptional process but are a separate functional state that renders the molecule incapable of further transcribing.
Explains Bustamante, "Our observations are consistent with the idea that a paused RNAP molecule is not just stopped there, like at a traffic light, but has parked inside a garage and must be brought out of the garage to resume its movement."
Bustamante and his colleagues speculate that within a cell there may be other molecules that bind to the RNAP to stabilize its conformation, so that it transcribes at a faster or slower rate-which in turn would make it more or less susceptible to pausing. This, they say, could be the basis for yet another level of transcriptional control and regulation. Future experiments are planned using an improved second-generation version of the integrated laser trap/flow control video microscope, which is now being built.
Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.
NOTE: Carlos Bustamante is a professor in UC Berkeley's Department of Molecular and Cell Biology, the Department of Physics, and the Department of Chemistry. He also heads the Advanced Microscopies Department for Berkeley Lab's Physical Biosciences Division. He is the coauthor of a paper on a single-molecule study of DNA polymerase in the March 2, 2000 issue of the journal Nature. (Wuite, et al, Vol. 404:p103-106) Reporters should contact him by e-mail at email@example.com.