Molecular anatomy of RNA polymerase using protein-conjugated metal probes with nuclease and protease activities

Abstract

Iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate (FeBABE) with the sequence-non-specific cleavage activity of nucleic acids and proteins was conjugated to protein Cys residues, and used for mapping the contact sites of both the α-subunit carboxy-terminal domain of Escherichia coli RNA polymerase on promoter UP elements and the σ⁷⁰ and σ³⁸ subunits on the respective promoters. The same chemical nuclease was also used as a chemical protease for mapping the subunit–subunit contact sites within the RNA polymerase. By using 2-iminothiolane as a linker, FeBABE could be conjugated to protein Lys residues and successfully used for mapping the contact surfaces of some E. coli transcription factors on the RNA polymerase holoenzyme.