RESUMO
Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Animais , Sequência de Bases , Biologia Computacional , Sequência Conservada , DNA/metabolismo , Evolução Molecular , Proteínas de Homeodomínio/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
A major class of bacterial small, noncoding RNAs (sRNAs) acts by base-pairing with mRNAs to alter the translation from and/or stability of the transcript. Our laboratory has shown that Hfq, the chaperone that mediates the interaction of many sRNAs with their targets, is required for the virulence of the enteropathogen Yersinia pseudotuberculosis. This finding suggests that sRNAs play a critical role in the regulation of virulence in this pathogen, but these sRNAs are not known. Using a deep sequencing approach, we identified the global set of sRNAs expressed in vitro by Y. pseudotuberculosis. Sequencing of RNA libraries from bacteria grown at 26 °C and 37 °C resulted in the identification of 150 unannotated sRNAs. The majority of these sRNAs are Yersinia specific, without orthologs in either Escherichia coli or Salmonella typhimurium. Six sRNAs are Y. pseudotuberculosis specific and are absent from the genome of the closely related species Yersinia pestis. We found that the expression of many sRNAs conserved between Y. pseudotuberculosis and Y. pestis differs in both timing and dependence on Hfq, suggesting evolutionary changes in posttranscriptional regulation between these species. Deletion of multiple sRNAs in Y. pseudotuberculosis leads to attenuation of the pathogen in a mouse model of yersiniosis, as does the inactivation in Y. pestis of a conserved, Yersinia-specific sRNA in a mouse model of pneumonic plague. Finally, we determined the regulon controlled by one of these sRNAs, revealing potential virulence determinants in Y. pseudotuberculosis that are regulated in a posttranscriptional manner.
Assuntos
RNA Bacteriano/genética , RNA não Traduzido/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Camundongos , Anotação de Sequência Molecular , Dados de Sequência Molecular , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Reprodutibilidade dos Testes , Especificidade da Espécie , Transcrição Gênica , Virulência/genética , Yersinia pestis/genética , Yersinia pestis/patogenicidade , Infecções por Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/microbiologiaRESUMO
MOTIVATION: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA-protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. RESULTS: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF-DNA recognition, and suggest a rational approach for future analyses of TF families.
Assuntos
Biologia Computacional/métodos , DNA/química , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA/metabolismo , Fatores de Transcrição/químicaRESUMO
In Drosophila melanogaster, the small interfering RNA (siRNA) pathway is triggered by exogenous double-stranded RNA (dsRNA) or upon viral infection. This pathway requires Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) called R2D2. A potentially distinct siRNA pathway, which requires Dcr-2 in association with a different dsRBP, called Loquacious (Loqs), is activated by endogenous dsRNA derived from transposons, structured loci and overlapping transcripts. Here we show that different sources of dsRNA enter a common siRNA pathway that requires R2D2 and Loqs. R2D2 and loqs mutants show impaired silencing triggered by injection of exogenous dsRNA or by artificial and natural expression of endogenous dsRNA. In addition, we show that these dsRBPs function sequentially and nonredundantly in collaboration with Dcr-2. Loqs is primarily required for dsRNA processing, whereas R2D2 is essential for the subsequent loading of siRNAs into effector Ago-RISC complexes.