Estudios in vivo E in vitro del tallo ribosómico de Saccharomyces cerevisiae mediante técnicas biofísicas y bioquímicas
AdvisorGarcía Ballesta, Juan Pedro
EntityUAM. Departamento de Biología Molecular
SubjectsRIbosomas - Análisis - Tesis doctorales; Biología y Biomedicina / Biología
NoteTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-12-2005
Summary The stalk is a vey dinamic and flexible estructure located in the large nbosomal subunit. The S. cerevisiae stalk is composed of four 12 kDa acidic proteins (Pla, PlP, P2a and P2P) forming a pentamer along with PO protein. There is a pool of acidic proteins fkee in the cytoplasm that can be exchanged with those bound to the nbosome. The structure and function of the staík remains quite unknown, although it seems to be involved in the interaction with soluble translation factors and, somehow, with the pattern of expressed proteins, depending on the different compositions it can adopt. Due to the flexibility of the stak, it is difficult to obtain models using X-ray difiaction or cryomicroscopy, so it is interesting to find some other different approaches to determine its structure and composition. In this work, chemical crosslinking, 2-photon microscopy coupled to fluorescence correlation spectroscopy (FCS) and electrophoretic techniques were used in order to know more about the number and distribution of the acidic proteins in the stalk. The cross-linking results determined that at least P2a protein binds to the nbosome mainly as a monomer. It was also observed that there is an interaction between P2a and PlP, which probably bind to the nbosome as an heterodimer. The FCS experiments allowed to determine more accurately the composition of the nbosomal stalk in vivo, finding that the binding of the acidic proteins to the ribosome is a very dynamic proccess, and that in the cells can coexist different nbosome subpopulations regarding their stalk composition. The most abundant population is composed by nbosomes carrying four different acidic proteins, but there are also minoritary subpopulatios in which the stalk can be composed by different combinations of homodimers and heterodimers. The comparison of these results with the ones obtained in vitro by electrophoresis &er the purification of the nbosomes showed that this latter technique greatly understimate the small heterogeneus nbosome subpopulations, and don't reflect the high dynamism that can be observed by FCS. Based on these and on previous results, we propose a model for the main nbosome subpopulation, in which the four different acidic proteins are present in the nbosomal staík. This model implies that the two P1 proteins are close to each other, the same as the P2 ones, and there is some assyrneúy in the stak, since the P2 proteins are extended to the cytoplasm, while the P1 proteins are bent over the nbosomal body.
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Google Scholar:García Marcos, Alberto
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