Estudio cristalográfico de proteínas implicadas en la ruta de los inositoles polifosfatados y su relación con el ARN

Los inositoles polifosfatados (IPs) son un amplio y diverso grupo de pequeñas moléculas solubles que desempeñan papeles biológicos en señalización celular y procesos metabólicos. La ruta de los IPs comienza con la síntesis de inositol 1,4,5-trisfosfato (IP3), un segundo mensajero muy conocido que promueve la liberación de Ca2+ de los sistemas de almacenamiento y regula su homeostasis. El precursor de IP3 es el inositol lipídico PIP2, el cual a su vez es sustrato de la enzima PI3K, uno de los principales efectores de la proteína reguladora de señalización celular RAS. Por tanto, RAS/PI3K participan en la regulación de los niveles de IP3. A partir del IP3, se forman el resto de IPs mediante la acción de diferentes quinasas y fosfatasas. Esta tesis se centra en el estudio de las inositol polifosfato quinasas (IPKs) de mamíferos, que regulan los niveles de IPs mediante la fosforilación del anillo de inositol en sus diferentes posiciones. En la última década, se ha puesto de manifiesto que muchos papeles de los IPs y las IPKs están directamente relacionados con eventos del ARN y con proteínas que lo reconocen. Este trabajo también se ha centrado en el análisis de una proteína implicada en la terminación de la transcripción en levaduras y a su vez relacionada con la señalización por RAS y otras rutas de IPs/IPKs. En cuanto a la biología estructural de las IPKs, se han estudiado dos familias diferentes de IPKs mediante Cristalografía de rayos X: la familia de inositol 1,3,4,5,6-pentaquisfosfato 2-quinasa (IP5 2-K) y la familia de inositol 1,2,3,4,5,6-hexaquisfosfato 6-quinasa (IP6K)..

The two kinases, AbrC1 and AbrC2, of the atypical two-component system AbrC are needed to regulate antibiotic production and differentiation in Streptomyces coelicolor

Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. gRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.We would like to thank Dr S. Buratowski for gratefully pro-viding yeast strains and plasmids. Data collection was per-formed at ESRF, ID23-1 beamline (Grenoble, France) and at ALBA Synchrotron, XALOC-BL13 beamline, with the collaboration of ESRF and ALBA staff.S