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Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

ABSTRACT: The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNA-binding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.

SUBMITTER: Liu Y

PROVIDER: S-EPMC1413715 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

Liu Yingfang Y Manna Adhar C AC Pan Cheol-Ho CH Kriksunov Irina A IA Thiel Daniel J DJ Cheung Ambrose L AL Zhang Gongyi G

Proceedings of the National Academy of Sciences of the United States of America 20060202 7

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual res ...[more]

PMID: 16455801

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Project description:SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.

Dataset Information

Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

Publications

Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

Similar Datasets

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