Affinity Isolation and I-DIRT Mass Spectrometric Analysis of the Escherichia coli O157:H7 Sakai RNA Polymerase Complex

Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the Escherichia coli K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic Escherichia coli strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although E. coli O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the “core ” E. coli genome. In all bacteria, one multisubunit RNA polymerase (RNAP) is responsible for the synthesis of RNA. In the widely studied model bacteria Escherichia coli K-12, the RNAP core enzyme is a complex of five subunits (2) that is competent for DNA-directed RNA synthesis but is reliant upon the binding of a dissociable sigma factor, which forms the holoenzyme, for promoter recognition and tran