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The packaging of DNA by histones is, in fact, so good that mobile, or motor, proteins that need access to DNA to do their jobs have a hard time getting space. For example, RNA polymerase, which constructs RNA by “reading” DNA strands, pauses or stalls entirely when it encounters a nucleosome in its path. HHMI investigator Michelle Wang of Cornell University in Ithaca, New York, is working to understand the mechanics of this process. “If DNA is really tightly packed in the nucleosome, then how do motor proteins, which carry out all the important functions, access the DNA that's buried in the nucleosome?” she asks.
For genes to be transcribed and expressed, DNA carrying the genetic message must temporarily unwind from the histone spools. Something must happen to the bonds between histones and the DNA to allow proteins like RNA polymerase to invade the nucleosome.
In 1997, nearly 25 years after the discovery of nucleosomes, Luger and her colleague Timothy Richmond, of the Swiss Federal Institute of Technology, in Zurich, solved the x-ray crystal structure of the nucleosome in atomic detail. From this discovery, they were able to make suppositions about the strengths of the bonds between histones and DNA, and where they occur. Luger suspected that locations where histones had fewer contact points with DNA required less energy to separate.
To evaluate those bonds, Wang and her colleagues started with Luger's x-ray crystal structure of a nucleosome. A molecule's crystal structure allows researchers to visualize contact points between DNA and histones but doesn't describe the strength of those bonds. In a February 2009 paper in Nature Structural & Molecular Biology, Wang and her colleagues describe experiments in which they mechanically “unzipped” double-stranded molecules of DNA one base pair at a time and created a quantitative map that details the locations and strengths of the DNA–histone interactions. They also measured the force required to release DNA from each histone carrier.
Says Luger, “X-ray crystallography can ‘see' the interactions, whereas single-molecule studies can ‘feel' them.”
Wang's measurements confirm Luger's predictions: fewer contacts between DNA and histones result in weaker interactions, but with one surprise. The crystal structure of the nucleosome led scientists to believe that an RNA polymerase pauses or stalls at three obstacles—strong DNA-histone interactions—along the nucleosome before DNA buried in the nucleosome becomes accessible for transcription (the process in which a DNA strand is transcribed into RNA). Wang's study implies that RNA polymerase may need to overcome only two of these barriers before the histones are ejected from the DNA, giving the polymerase “a free ride, as if it moves on naked DNA.” Wang's group and several others are investigating whether this roadmap of barriers dictates which genes will be transcribed.
Photo: Kevin Rivoli / AP ©HHMI