We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase σ70 subunit. Trypsin-resistant fragments containing σ70 conserved region 2 (σ702), and carboxy-terminal fragments containing conserved regions 3 and 4 (σ703-4) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of σ70·σ702 bound core RNA polymerase competitively with intact σ70. In contrast to σ702 alone, the RNA polymerase holoenzyme formed with σ702 specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). σ702 also forms crystals that are suitable for X-ray analysis. σ703-4 bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on σ70 was further localized to within σ70[551-608], comprising σ conserved region 4.2.