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Science at the Frontier: Volume I
by trial and error, that eventually match the profile of the protein they are trying to isolate and purify. These DNA affinity columns are then attached to a solid substrate and put into a chemical solution. When a solution with thousands of candidate proteins is washed past these tethered DNA strings that geneticists refer to as binding sites, the targeted transcription factor recognizes its inherent binding sequence and chemically hooks on to the probe. Once the transcription factor is in hand, it can be analyzed and duplicated, often by as much as a factor of 105 in a fairly reasonable amount of laboratory time.
Tjian illustrated another method of doing binding studies, that is, isolating the small region of the DNA sequence that actually contacts and binds the transcription factor. The first step is to tag the DNA gene region by radioactive labeling and then to send in the transcription factor to bind. Next, one tries to probe the connection with "attacking agents, small chemicals or an enzyme that cuts DNA." The bound protein literally protects a specific region of the DNA from chemical attack by these agents and thus allows the detailed mapping of the recognition site. "Gel electrophoresis patterns can actually tell, to the nucleotide, where the protein is interacting" (Figure 5.5).
After some years now of experience with binding studies, Tjian and other molecular biologists have begun to recognize certain signatures , structures that seem to indicate transcription factor binding domains. One of the most prominent are the so-called zinc fingers, actually a specific grouping of amino acids that contains a zinc molecule located between cysteine and histidine residues. Over and over again in binding studies, analysis of complex proteins showed Tjian this "recognizable signpost . . . a zinc finger," which, he and his colleagues surmised, "very likely binds DNA." Subsequent analysis showed that it was, in fact, usually embedded in the effective binding domain. In this very specialized area of research, Tjian called this discovery "an extremely powerful piece of information" that has led to the cataloging of a large family of so-called zinc-finger binding proteins. Another similar binding signature they call a helix-turn-helix, or a ''homeodomain.''
Using the Power of Genetics to Study Transcription
Biochemist Kevin Struhl described for symposium participants some of the various methods used in his laboratory's work with yeast, an organism whose relative simplicity and rapid reproducibility make it a good candidate for studies of the transcription process. "As it