The pre-SIi variants defective for open complex formation fail to interact with σ54.
A. Top: Schematic and nucleotide sequence of the S. meliloti nifH mismatch promoter probe, the lowercase letters in bold type-face indicate non-complementary residues in the mismatch promoter probe. Bottom: SDS-PAGE gel showing the cross-linking profiles of σ54–DNA complexes formed on the mismatch promoter probe in the presence of 4 mM dATP and either PspF1–275WT or variants. The migration positions of the cross-linked σ54–DNA and PspF1–275–DNA species are indicated. Native-PAGE gel illustrating supershift complexes (σ54ss–DNA) are formed in the presence of PspF1–275WT (lane 2) and the S135A (lane 6), Q136A (lane 7) and P137A/T (lanes 8 and 9) variants. The migration positions of the supershift (σ54ss–DNA) and binary σ54–DNA (σ54–DNA) complexes, free DNA and percentage DNA bound in each complex is indicated.
B. SDS-PAGE gel as in A but in the presence of core RNAP. The migration positions of the cross-linked β/β′–DNA and σ54–DNA species are indicated. Cross-linked β/β′–DNA species are only observed with the transcription competent PspF1–275 variants (WT, Q136A and P137A/T).