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New Microscopy Technique

NSF-Funded Researcher Uses New Microscopy Technique to Discover How Cells Synthesize Proteins

From the National Science Foundation

In a cell, DNA contained in genes directs the synthesis of specific proteins that determine the function of that cell. These functions have to be turned on and off in a precise manner for the cell to operate normally. This control of genes involves many complex interactions between proteins and DNA.

Carlos Bustamante, Ph.D., a National Science Foundation-supported researcher at the Institute of Molecular Biology at the University of Oregon, has reported in the journal Science recently the results of studies that reveal how this happens. He described how he used Scanning Force Microscopy (SFM), a new technique to visualize DNA and protein complexes.

Proteins bind to specific regions of DNA to inhibit expression of one or more genes. These proteins have to locate specific DNA target sites in a vast excess of non-specific DNA. To do this, they must have an efficient mechanism for recognition, and must discriminate between non-specific and specific sites by binding more tightly to the latter.

Recognition of the specific target site is often accompanied by DNA bending; however, the importance of this bending is not fully understood. SFM can reveal the extent of DNA bending induced by protein binding to non-specific and specific sites. SFM works like a phonograph, scanning a surface underneath a tip and sensing the deflections of the tip. These deflections can be amplified and recorded to provide a topographic map of the surface.

Bustamante has studied the binding of Cro, a protein that regulates genes in bacterial viruses, to DNA containing non-specific and specific binding sites. Cro binds loosely to DNA at non-specific sites, and travels along the chain until it recognizes a specific target site, where it binds very tightly. Binding, both non-specific and specific, is accompanied by bending of the DNA chain. SFM revealed an increase in the DNA bending angle where the protein is bound to a specific site.

Bustamante proposes that Cro first binds loosely and non-specifically to the DNA, then produces a bending wave as it travels along the strand. In order to recognize a specific site, the DNA must bend to allow for specific contacts with the protein. At a specific target site, the DNA is more easily bent, and this allows the protein access to specific contacts. Because Cro bends the non-specific sites, it must produce a bending wave as it travels along the strand.

Molecular recognition is important not only in gene regulation, but in many other biological processes, says Kamal Shukla, Molecular Biophysics Program Director at the NSF. It also has many applications in biotechnology. Visualizing this recognition illustrates the power of advanced biophysical techniques. SFM makes possible the characterization of many molecular assemblies that are too large to be determined by conventional X-ray crystallography and nuclear magnetic resonance techniques.


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