Mutations in the 1.1 subdomain of <em>Escherichia coli</em> sigma factor δ70 and disruption of its overall structure.

Among various group 1 sigma factors, two amino acids. Va155 and A1a59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]σ70 and [Gly59)σ70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type σ70 has an apparent molecular mass of 87 kDa. However, [Gly434]σ70 which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]σ70 and [Gly59]σ70 have reduced helicity (20%) compared to wild-type σ70 (50%). Binding of sigma factors with the hydrophobic, surface active probe I-anilinonapthalene-8-sulphonate, has shown that more hydrophobic suites are available/exposed in [Gly55]σ70. [Gly59]σ70 as well as in [Gly434]σ70 in comparison to wild-type σ70. Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (α2ββ&#39;) have shown reduced trunscnptional activity in comparison to the enzyme containing wild-type sigma factor. However; another mutation (Val→Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83] σ70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in σ70 may interact hydrophobically with the 2.3/2.4 DNA-binding region. [ABSTRACT FROM AUTHOR]