RESUMEN
RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.
Asunto(s)
Escherichia coli/enzimología , Regulación Enzimológica de la Expresión Génica , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Sitio Alostérico , Ácido Cítrico/química , Clonación Molecular , Cristalografía por Rayos X/métodos , Eliminación de Gen , Metabolómica/métodos , Metales/química , Modelos Químicos , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Polímeros/química , Unión ProteicaRESUMEN
In Escherichia coli and many other bacterial species, the glycolytic enzyme enolase is a component of the multi-enzyme RNA degradosome, an assembly that is involved in RNA processing and degradation. Enolase is recruited into the degradosome through interactions with a small recognition motif located within the degradosome-scaffolding domain of RNase E. Here, the crystal structure of enolase bound to its cognate site from RNase E (residues 823-850) at 1.9 A resolution is presented. The structure suggests that enolase may help to organize an adjacent conserved RNA-binding motif in RNase E.
Asunto(s)
Endorribonucleasas/química , Escherichia coli/enzimología , Fosfopiruvato Hidratasa/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Endorribonucleasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Alineación de SecuenciaRESUMEN
In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucina/metabolismo , Leucemia/patología , Proteínas de Fusión Oncogénica/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Transformación Celular Neoplásica , Femenino , Proteínas de Homeodominio/química , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Células Mieloides/citología , Células 3T3 NIH , Proteínas de Fusión Oncogénica/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel beta-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.
Asunto(s)
Biocatálisis , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Manganeso/química , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/química , ARN Helicasas/metabolismo , ARN Bacteriano/química , Secuencia de Aminoácidos , Calorimetría , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , ARN Helicasas/químicaRESUMEN
The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.