Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.
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.
Baxter
Kimberly
K
Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-0830, USA.
Lee
Jennifer
J
Minakhin
Leonid
L
Severinov
Konstantin
K
Hinton
Deborah M
DM
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics*; D001886:Bordetella pertussis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
C078876:AsiA protein, Enterobacteria phage T4; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C067996:MotA protein, Enterobacteria phage T4; D014157:Transcription Factors; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D000409:Alanine