The sorcerer's apprentice started a water-carrying system, but couldn't stop it, and soon he was up to his neck in water, and trouble. Living cells have a better design: When they activate a gene, they have a system in reserve to turn it off. The cell does not want to waste energy making proteins it no longer needs. Cornell researchers have identified two mechanisms cells use and found they are designed to be quick.

Peng Chen, associate professor of chemistry and chemical biology, and colleagues report the discovery in the online edition of the Proceedings of the National Academy of Sciences Sept. 4. "The generic is for the activator to fall off, then the repressor binds," Chen said. "What we have found is two pathways that are much more efficient."

The work was done in bacteria and could lead to new ways to kill harmful bacteria, Chen said. It also represents a step forward in understanding gene transcription regulation that could apply further up the evolutionary ladder.

The biochemical processes involved take place on the scale of single molecules, far too small to observe through a microscope. To monitor these processes, the researchers tagged proteins and DNA binding sites with molecules that fluoresce and change their fluorescence intensity when they meet, so that a flash of light appears or changes when a biochemical reaction takes place. The fluorescence change is triggered by a rearrangement of electric charges as the tagging molecules come together, beginning when they are near (a matter of nanometers) and becoming brighter as they close in. By tagging one end of a protein molecule, the researchers can tell by the brightness of the signal which orientation the protein takes in binding to DNA.

The researchers worked with bacteria for which copper is toxic. These bacteria possess genes that code for a protein that grabs copper atoms and shoves them out through the cell wall. Ordinarily a protein called CueR binds to the chromosome in front of the sites of these genes and distorts the DNA, preventing the gene from being transcribed. When copper atoms bind to CueR it changes its configuration, allowing enzymes to transcribe the genes to make the protective proteins.

When the copper threat is gone another form of CueR, circulating in solution in the cell, replaces the copper-modified form, turning off the gene. By watching and timing the reactions, the Cornell researchers determined that the turnoff protein either shoves the bound protein out of the way or "assists" it in detaching and then moves into the vacant space. These mechanisms are up to 70 times faster than waiting for the activator to go away on its own, the research showed. It is likely that bacteria use similar methods for turning off protection against other toxic metals, they said.

The study also showed that the CueR protein can bind to the DNA in two different orientations and can spontaneously flip between them without completely detaching. In one orientation it binds only at the specific gene it is supposed to control. In the other it attaches nonspecifically to DNA. The researchers suggest that this may make it easier for the protein to get to where it's needed quickly, by attaching to the DNA strand and sliding along until it finds the specific site, then flipping into working mode.

The research was supported primarily by the National Institutes of Health and partly by the National Science Foundation.