Use of site-specifically tethered chemical nucleases to study macromolecular reactionsReport as inadecuate

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Biological Procedures Online

, Volume 5, Issue 1, pp 78–89

Received: 19 November 2002Revised: 17 February 2003Accepted: 19 February 2003DOI: 10.1251-bpo49

Cite this article as: Mukherjee, S. & Sousa, R. Biol. Proced. Online 2003 5: 78. doi:10.1251-bpo49


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.

Indexing termsT7 RNA Polymerase Transcription Protein Conformational Changes Chemical Nucleases Fe-BABE Published: March 24, 2003

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Author: Srabani Mukherjee - Rui Sousa


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