Mutations in the 1.1 subdomain of Escherichia coli sigma factor σ70 and disruption of its overall structure

Gopal, Vijaya ; Chatterji, Dipankar (1997) Mutations in the 1.1 subdomain of Escherichia coli sigma factor σ70 and disruption of its overall structure European Journal of Biochemistry, 244 (2). pp. 613-618. ISSN 0014-2956

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Among various group I sigma factors, two amino acids, Val55 and Ala59 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]σ 70 and [Gly59]σ70 have reduced helicity (20%) compared to wild-type σ70 (50%). Binding of sigma factors with the hydrophobic, surface active probe 1-anilinonapthalene-8-sulphonate, has shown that more hydrophobic surfaces 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 (α2β β') have shown reduced transcriptional 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.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons, Inc..
Keywords:Sigma Factor (σ70); 1.1 Subdomain; DNA Binding; Conformation; Gel Mobility; Transcription Inhibition
ID Code:6298
Deposited On:20 Oct 2010 11:14
Last Modified:30 Sep 2011 10:06

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