Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma70 and disruption of its overall structure.
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]sigma70 and [Gly59]sigma70, 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 sigma70 has an apparent molecular mass of 87 kDa. However, [Gly434]sigma70, 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]sigma70 and [Gly59]sigma70 have reduced helicity (20%) compared to wild-type sigma70 (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]sigma70, [Gly59]sigma70 as well as in [Gly434]sigma70 in comparison to wild-type sigma70. 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 (alpha2beta beta') 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]sigma70, 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 sigma70 may interact hydrophobically with the 2.3/2.4 DNA-binding region.
Gopal
V
V
Centre for Cellular and Molecular Biology, Hyderabad, India.
Chatterji
D
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D015394:Molecular Structure; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases