Open Access

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

Biological Procedures Online5:51078

DOI: 10.1251/bpo49

Received: 19 November 2002

Accepted: 19 February 2003


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 terms

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