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

Srabani Mukherjee, Rui Sousa

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)78-89
Number of pages12
JournalBiological Procedures Online
Volume5
Issue number1
DOIs
StatePublished - 2003

Keywords

  • Chemical nucleases
  • Fe-BABE
  • Protein conformational changes
  • T7 RNA polymerase
  • Transcription

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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