Domain organization of the Escherichia coli RNA polymerase sigma(70) subunit

We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma(70) subunit. Trypsin-resistant fragments containing sigma(70) conserved region 2 (sigma(2)(70)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma(3-4)(70)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma(70).sigma(2)(70) bound core RNA polymerase competitively with intact sigma(70). In contrast to sigma(2)(70) alone, the RNA polymerase holoenzyme formed with sigma(2)(70) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). sigma(2)(70) also forms crystals that are suitable for X-ray analysis. sigma(3-4)(70) bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on sigma(70) was further localized to within sigma(70)[551-608], comprising sigma conserved region 4.2. (C) 1996 Academic Press Limited