RESUMEN
The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.
Asunto(s)
Bacillus subtilis/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Manganeso/metabolismo , Proteínas de Transporte de Membrana/genética , Secuencias Reguladoras de Ácido Ribonucleico , Riboswitch/genética , Regiones no Traducidas 5' , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Manganeso/farmacología , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Bacteriano/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Synthesis of the 31-amino acid, inner membrane protein MgtS (formerly denoted YneM) is induced by very low Mg2+ in a PhoPQ-dependent manner in Escherichia coli Here we report that MgtS acts to increase intracellular Mg2+ levels and maintain cell integrity upon Mg2+ depletion. Upon development of a functional tagged derivative of MgtS, we found that MgtS interacts with MgtA to increase the levels of this P-type ATPase Mg2+ transporter under Mg2+-limiting conditions. Correspondingly, the effects of MgtS upon Mg2+ limitation are lost in a ∆mgtA mutant, and MgtA overexpression can suppress the ∆mgtS phenotype. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly controlled Mg2+ transporter and adds to the list of small proteins that regulate inner membrane transporters.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/genética , ATPasas Tipo P/metabolismoRESUMEN
The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/genética , ARN Bacteriano/genética , Bacillus subtilis/metabolismo , Eliminación de Gen , Proteína de Factor 1 del Huésped/análisis , Proteína de Factor 1 del Huésped/metabolismo , Sistemas de Lectura Abierta , ARN sin Sentido/análisis , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Bacteriano/análisis , ARN Bacteriano/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Metabolite-sensing regulatory RNAs, oft referred to as riboswitches, are widely used among eubacteria for control of diverse biochemical pathways and transport mechanisms. Great strides have been made in understanding the general structure and biochemistry of individual riboswitch classes. However, along with these advancements, it has become clear that metabolite-sensing riboswitches respond to an increasingly structurally diverse range of metabolite and metal ligands. Moreover, the recent accruement of new riboswitches has uncovered individual examples and classes that utilize unique regulatory strategies or employ a regulatory logic other than simple feedback inhibition.
Asunto(s)
ARN Bacteriano/fisiología , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Conformación de Ácido Nucleico , ARN Bacteriano/químicaRESUMEN
Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.
Asunto(s)
Nucléolo Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Proteínas de Unión al ARN/metabolismo , Proteínas ras/biosíntesis , Células Cultivadas , Humanos , Señales de Localización Nuclear , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/metabolismo , NucleolinaRESUMEN
Tuberin (TSC2) is a tumor suppressor gene. At the cellular level, tuberin is required as a critical regulator of cell growth, neuronal differentiation, and tumor suppression. Here we report a critical role for tuberin in late stage myeloid cell differentiation. Tuberin strongly augments transforming growth factor (TGF)-beta1 signal transduction pathways, including SMAD activation. We also demonstrate that the amino-terminal region of tuberin interacts specifically with the MH2 domain of SMAD2 and SMAD3 proteins to regulate TGF-beta1-responsive genes such as p21(CIP). Inhibition of tuberin expression by Tsc2 antisense greatly reduces the ability of TGF-beta to transcriptionally regulate p21(CIP), p27(KIP), and cyclin A leading to an abrogation of the antiproliferative effects of TGF-beta1. Also, inhibition of tuberin expression during stimulation of monocytic differentiation with vitamin D(3) and TGF-beta1 significantly impaired myeloid cell growth inhibition and differentiation. Together, the data demonstrate the presence of a novel activation process following TGF-beta1 stimulation that requires tuberin-dependent activity.