You may think you don't have much in common with those nasty little bacteria that reside in disgusting locales such as spoiled meat or raw sewage, but think again. Surprisingly, humans and the Escherichia coli (E. coli) bacterium, an inhabitant of the intestinal tract, share over 2,000 similar genes. Most of these genes are so essential for life on earth that they were passed down eons ago by a common evolutionary ancestor of both humans and bacteria. This genetic kinship and the ease of cultivating bacteria in the laboratory have made E. coli an ideal model organism for studying genetics. L. James Maher, III, Ph.D., of the Mayo Clinic Department of Biochemistry and Molecular Biology, has taken just such an approach to study genetic "switches" that turn genes "on" or "off" in living organisms. According to Maher, "For 50 years now, scientists have turned to the lowly bacterium to understand basic genetic mechanisms that are similar in organisms as diverse as E. coli and humans."

Studying DNA flexibility in bacteria

Maher and colleagues, Nicole A. Becker, Ph.D., also of Mayo, and collaborator Dr. Jason D. Kahn of the University of Maryland, used a bacterial model system to obtain new insights into how DNA flexibility can be altered in cells to turn genes "on" or "off" (J. Mol. Biol. 349, 716-730). Since the 1950s, scientists have studied a bacterial gene group known as the lac operon that now serves as a model for understanding gene regulation. Flipping a molecular switch to turn lac operon genes "off" requires DNA flexibility. This is because two sites that are distant from each other on the string of DNA must be brought close together, requiring DNA looping. Maher and colleagues were interested in a type of long-range DNA looping that occurs in cells. Such looping controls whether or not the code information in each gene is used to make the corresponding protein product that performs useful functions in the cell. This type of looping, known as "action at a distance," brings two distant DNA sites in close proximity to each other. The resulting DNA loop can turn a gene "off" or "on."

Why is DNA flexibility important? To illustrate structural properties of the DNA double helix to students, Maher uses a short stretch of garden hose scavenged from his back yard. Like a garden hose, DNA is a linear, roughly cylindrical structure that is flexible enough to bend, and in some instances, form loops. However, like the garden hose, the flexibility of DNA has limits. Maher demonstrates that a minimum length of hose—and by analogy, DNA—is needed to form a loop. Try forming a loop using a segment of hose shorter than this minimal length, and the inherent rigidity of the hose prevents looping. For years scientists have been puzzled by a curious observation. Explains Kahn, "When we study DNA in the test tube, it's very stiff, but when we look at in cells, it acts as if it's much more flexible. So we wanted to use bacteria as a type of 'living test tube' to investigate DNA flexibility in cells." Researchers wondered what tricks a bacterial cell can use to form DNA loops smaller than those capable of forming in solution, a feat that has important consequences for our understanding of gene regulation in living organisms.

Proteins Can Increase DNA Flexibility in Cells

To answer this question, Maher's group devised a genetically engineered E. coli strain to study DNA looping inside living cells. Using a modified version of the well-studied lac operon system, researchers systematically examined the dependence of DNA loop formation upon the DNA length and alignment between two distant DNA sites bound by the lac repressor protein. For the lac repressor protein to contact both DNA sites simultaneously, DNA looping is required. Varying the length of DNA between the sites, they were able to compare the ability of the lac repressor to turn "off" a reporter gene whose protein product could be easily detected. In agreement with previous studies, the researchers found that in the cell, the alignment of the two sites was much more important than the length of DNA separating the sites, suggesting that the cell had found ways to reduce the minimal DNA length required for looping of DNA in the test tube. Maher and colleagues wondered whether the cell might use proteins to increase the flexibility of DNA. A candidate protein known to bend DNA in E. coli is called HU. Maher hypothesized that if E. coli cells were genetically engineered to lack HU, then DNA bending, and by extension looping, might be compromised. This theory proved correct, and small DNA loops were much less common in the absence of HU protein.

Taking this finding one step further, Maher and colleagues showed that a DNA-bending protein (HMGB1) from mammals could also increase the flexibility of DNA so that small DNA loops could form in bacteria. Returning to the hose analogy, the HU and HMG proteins might act as pliers that produce sharp kinks in the hose, allowing smaller loops to form than are possible in their absence. Taken together, Maher's findings are the first to definitively explain why DNA is more flexible in the cell than in the test tube. In addition, the fact that a mammalian HMG protein could substitute for the bacterial HU protein to bend E. coli DNA suggests that similar mechanisms might increase DNA flexibility in human cells.

Implications

How do these findings enhance our understanding of gene regulation? According to Maher, "We now know that the intrinsic physical properties of DNA are not well-suited to function in the molecular switches required for the regulation of some genes. In these cases, DNA rigidity must be altered by helper proteins that increase DNA flexibility." He envisions further applications of this engineered bacterial system, for example, examining the DNA-bending ability of mutated HU and HMG proteins to determine which regions of the proteins are important for their functions, and using the system to test other suspected DNA-bending proteins. Defects in DNA-bending proteins are responsible for certain human diseases. For example, some patients with a genetic disease called Turner syndrome have mutations in a DNA-bending protein called SRY. A member of the HMG family of proteins, SRY is important for testis development in the human fetus. When the gene for SRY is mutated, the testes do not develop properly. Individuals with this form of Turner syndrome, although genetically male, exhibit female physical characteristics and have various abnormalities such as short stature, lack of sexual development at puberty, and infertility. Scientists have discovered mutations in the DNA-bending region of SRY that affect its function in the cells of Turner syndrome patients.

In the future, scientists might be able to use what they've learned about the importance of DNA bending to genetically engineer useful DNA-bending proteins. Such artificial proteins could be made to bend specific genetic sequences to turn disease-related genes "on" or "off." "My lab is trying to engineer an artificial DNA-looping protein," comments Kahn. "We will probably use the bacterial system developed in Dr. Maher's lab to assess the new protein's properties." Maher and Kahn stress that the clinical use of artificial DNA-bending proteins is still remote on the gene therapy horizon. Nevertheless, studies on genetic switches in the lowly bacterium help bring scientists one step closer to understanding how genes are controlled in humans, and how they could perhaps be artificially manipulated to treat human disease.