NDLI: Mutational analysis of σ70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA

Content Provider	PubMed Central
Author	Baxter, Kimberly Lee, Jennifer Minakhin, Leonid Konstantin, Severinov Hinton, Deborah M.
Abstract	Transcriptional activation of T4 middle promoters requires σ70-containing E. coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the σ70 –10 DNA element. However, these promoters lack the σ70 –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 σ70, the C-terminal portion that normally contacts –35 DNA and the β-flap structure in core. AsiA binding prevents the σ70/β-flap and σ70/-35 DNA interactions, inhibiting transcription from promoters that require a –35 element. To test the importance of residues within σ70 region 4 for MotA and AsiA function, we investigated how σ70 region 4 mutants interact with AsiA, MotA, and the β-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 β-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/β-flap interaction, significantly impair the AsiA/σ70 interaction, and eliminate AsiA inhibition. However, the 4.1 mutant σ70 is still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to σ70 region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the σ70 C-terminus. Our analyses provide biochemical relevance for the σ70 residues identified in the structure, indicate that the interaction of AsiA with σ70 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 σ70.
Related Links	http://dx.doi.org/10.1016/j.jmb.2006.08.074
Ending Page	944
Page Count	14
Starting Page	931
File Format	PDF
ISSN	00222836
e-ISSN	10898638
Journal	Journal of molecular biology
Issue Number	5
Volume Number	363
Language	English
Publisher Date	2006-11-01
Access Restriction	Open
Subject Keyword	Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Structural Biology Molecular Biology Biophysics

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The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70 Subunit of Escherichia coli RNA Polymerase

Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle

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Substitutions in Bacteriophage T4 AsiA and Escherichia coli ς70 That Suppress T4 motA Activation Mutations

Bacteriophage T4 MotA Activator and the β-Flap Tip of RNA Polymerase Target the Same Set of σ70 Carboxyl-terminal Residues*

Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters.

Mutational analysis of σ70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA

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