Crosslinking mass spectrometry was applied to investigate the localization of σR1.1 domain in the structure of Spirochaeta africana RNA polymerase holoenzyme. RNA polymerase holoenzymes were assembled using polymerase core subunits and σ factor heterologously expressed in Escherichia coli. Two complexes were assembled in the absence of S. africana CarD activator and two in the presence of CarD activator. The resulting four complexes were crosslinked using disuccinimidyl sulfoxide (DSSO). Crosslinked protein complexes were analyzed by LC-MS/MS, and crosslinked residue pairs were identified in the form of crosslinked peptides.