PMID:16996538

Citation	No author listed (2006) Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA. J. Mol. Biol. 363:931-44
Abstract	Transcriptional activation of bacteriophage T4 middle promoters requires sigma70-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the sigma70 -10 DNA element. However, these promoters lack the sigma70 -35 element, having instead a MotA box centered at -30, which is bound by MotA. Previous work has indicated that AsiA and MotA interact with region 4 of sigma70, the C-terminal portion that normally contacts -35 DNA and the beta-flap structure in core. AsiA binding prevents the sigma70/beta-flap and sigma70/-35 DNA interactions, inhibiting transcription from promoters that require a -35 element. To test the importance of residues within sigma70 region 4 for MotA and AsiA function, we investigated how sigma70 region 4 mutants interact with AsiA, MotA, and the beta-flap and function in transcription assays in vitro. We find that alanine substitutions at residues 584-588 (region 4.2) do not impair the interaction of region 4 with the beta-flap or MotA, but they eliminate the interaction with AsiA and prevent AsiA inhibition and MotA/AsiA activation. In contrast, alanine substitutions at 551-552, 554-555 (region 4.1) eliminate the region 4/beta-flap interaction, significantly impair the AsiA/sigma70 interaction, and eliminate AsiA inhibition. However, the 4.1 mutant sigma70 is still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to sigma70 region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the sigma70 C terminus. Our analyses provide biochemical relevance for the sigma70 residues identified in the structure, indicate that the interaction of AsiA with sigma70 region 4.2 is crucial for activation, and support the idea that AsiA binding facilitates an interaction between MotA and the far C terminus of sigma70.
Links	PubMed PMC1698951 Online version:10.1016/j.jmb.2006.08.074
Keywords	Alanine/genetics; Amino Acid Sequence; Bacteriophage T4/genetics; Bordetella pertussis/genetics; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Directed RNA Polymerases/chemistry; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Models, Molecular; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Conformation; Transcription Factors/chemistry; Transcription Factors/genetics; Transcriptional Activation; Viral Proteins/chemistry; Viral Proteins/genetics
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
edit table

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.