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Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.

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.

SUBMITTER: Baxter K

PROVIDER: S-EPMC1698951 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.

Baxter Kimberly K Lee Jennifer J Minakhin Leonid L Severinov Konstantin K Hinton Deborah M DM

Journal of molecular biology 20060830 5

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 ...[more]

PMID: 16996538

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Project description:Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth.

Dataset Information

Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.

Publications

Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.

Similar Datasets

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