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Use of site-specifically tethered chemical nucleases to study macromolecular reactions

Abstract

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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Correspondence to Rui Sousa.

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Published: March 24, 2003

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Mukherjee, S., Sousa, R. Use of site-specifically tethered chemical nucleases to study macromolecular reactions. Biol. Proced. Online 5, 78–89 (2003). https://doi.org/10.1251/bpo49

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Indexing terms

  • T7 RNA Polymerase
  • Transcription
  • Protein Conformational Changes
  • Chemical Nucleases
  • Fe-BABE