RESUMEN
Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.
Asunto(s)
Lípidos/biosíntesis , Mycobacterium tuberculosis/enzimología , Sulfotransferasas/química , Sitios de Unión , Cromatografía en Capa Delgada , Cristalografía por Rayos X , Citosol/enzimología , Bases de Datos como Asunto , Densitometría , Dimerización , Disacáridos/química , Enlace de Hidrógeno , Cinética , Lípidos/química , Espectrometría de Masas , Modelos Químicos , Modelos Moleculares , Mycobacterium/enzimología , Mycobacterium smegmatis/metabolismo , Sistemas de Lectura Abierta , Oxígeno/química , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Serina/química , Relación Estructura-Actividad , Transgenes , Trehalosa/química , Rayos XRESUMEN
We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.
Asunto(s)
ADN Complementario/genética , Hepacivirus/genética , Péptido Hidrolasas/metabolismo , Péptidos/genética , Fosfotransferasas/metabolismo , Proteómica/métodos , ADN Complementario/metabolismo , Análisis por Micromatrices/métodos , Péptidos/metabolismo , Fosforilación , Especificidad por Sustrato , Proteínas no Estructurales Virales/metabolismoRESUMEN
Translation, storage, and degradation of messenger ribonucleic acids (mRNAs) are key steps in the posttranscriptional control of gene expression, but how mRNAs transit between these processes remains poorly understood. In this paper, we functionally characterized the DExD/H box adenosine triphosphatase (ATPase) Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, we showed that Dhh1 was sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression was followed by mRNA decay; however, deleting components of the 5'-3' decay pathway uncoupled these processes. Whereas Dhh1's ATPase activity was not required to induce translational inhibition and mRNA decay when directly tethered to an mRNA, ATP hydrolysis regulated processing body dynamics and the release of Dhh1 from these RNA-protein granules. Our results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored, or decayed.
Asunto(s)
Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/metabolismo , Regulación Fúngica de la Expresión Génica , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ARN Helicasas DEAD-box/genética , Fructosa-Bifosfato Aldolasa/biosíntesis , Fructosa-Bifosfato Aldolasa/genética , Hidrólisis , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Factores de TiempoRESUMEN
RNA levels are determined by the rates of both transcription and decay, and a mechanistic understanding of the complex networks regulating gene expression requires methods that allow dynamic measurements of transcription and decay in living cells with minimal perturbation. Here, we describe a metabolic pulse-chase labeling protocol using 4-thiouracil combined with large-scale RNA sequencing to determine decay rates of all mRNAs in Saccharomyces cerevisiae. Profiling in various growth and stress conditions reveals that mRNA turnover is highly regulated both for specific groups of transcripts and at the system-wide level. For example, acute glucose starvation induces global mRNA stabilization but increases the degradation of all 132 detected ribosomal protein mRNAs. This effect is transient and can be mimicked by inhibiting the target-of-rapamycin kinase. Half-lives of mRNAs critical for galactose (GAL) metabolism are also highly sensitive to changes in carbon source. The fast reduction of GAL transcripts in glucose requires their dramatically enhanced turnover, highlighting the importance of mRNA decay in the control of gene expression. The approach described here provides a general platform for the global analysis of mRNA turnover and transcription and can be applied to dissect gene expression programs in a wide range of organisms and conditions.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Estabilidad del ARN/genética , ARN de Hongos/metabolismo , ARN Mensajero , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Galactosa/metabolismo , Biblioteca de Genes , Genoma Fúngico , Estudio de Asociación del Genoma Completo , Glucosa/deficiencia , Glucosa/genética , Semivida , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ARN , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Tiouracilo/análogos & derivados , Tiouracilo/análisis , Tiouracilo/metabolismo , Transcripción GenéticaRESUMEN
Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Levaduras/metabolismo , Transporte Activo de Núcleo Celular , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteína I de Unión a Poli(A)/deficiencia , Proteína I de Unión a Poli(A)/genética , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Levaduras/genética , Proteína Exportina 1RESUMEN
The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.