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In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

ABSTRACT: The small RNAs CsrB and CsrC of Salmonella indirectly control the expression of numerous genes encoding widespread cellular functions, including virulence. The expression of csrB and csrC genes, which are located in different chromosomal regions, is coordinated by positive transcriptional control mediated by the two-component regulatory system BarA/SirA. Here, we identified by computational analysis an 18-bp inverted repeat (IR) sequence located far upstream from the promoter of Salmonella enterica serovar Typhimurium csrB and csrC genes. Deletion analysis and site-directed mutagenesis of the csrB and csrC regulatory regions revealed that this IR sequence is required for transcriptional activation of both genes. Protein-DNA and protein-protein interaction assays showed that the response regulator SirA specifically binds to the IR sequence and provide evidence that SirA acts as a dimer. Interestingly, whereas the IR sequence was essential for the SirA-mediated expression of csrB, our results revealed that SirA controls the expression of csrC not only by binding to the IR sequence but also by an indirect mode involving the Csr system. Additional computational, biochemical, and genetic analyses demonstrated that the integration host factor (IHF) global regulator positively controls the expression of csrB, but not of csrC, by interacting with a sequence located between the promoter and the SirA-binding site. These findings contribute to the better understanding of the regulatory mechanism controlling the expression of CsrB and CsrC.

SUBMITTER: Martinez LC

PROVIDER: S-EPMC3911252 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

Martínez Luary C LC Martínez-Flores Irma I Salgado Heladia H Fernández-Mora Marcos M Medina-Rivera Alejandra A Puente José L JL Collado-Vides Julio J Bustamante Víctor H VH

Journal of bacteriology 20131101 2

The small RNAs CsrB and CsrC of Salmonella indirectly control the expression of numerous genes encoding widespread cellular functions, including virulence. The expression of csrB and csrC genes, which are located in different chromosomal regions, is coordinated by positive transcriptional control mediated by the two-component regulatory system BarA/SirA. Here, we identified by computational analysis an 18-bp inverted repeat (IR) sequence located far upstream from the promoter of Salmonella enter ...[more]

PMID: 24187088

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Project description:BackgroundWith the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (approximately 90% AT) presents significant challenges to in silico cis-regulatory element discovery.ResultsWe have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.ConclusionThis GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

Dataset Information

In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

Publications

In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

Similar Datasets

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