Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits. Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3. This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex.