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Biol Proced Online. 2003;5:78-89. Epub 2003 Mar 24.

Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions.

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Department of Biochemistry, University of Texas Health Science Center at San Antonio. 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. USA.


During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP). Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches.


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