RESUMEN
The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfoproteínas/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/fisiología , Saccharomyces cerevisiae/genéticaRESUMEN
The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein PO. This complex interacts at the GTPase domain in the large subunit rRNA, overlapping the binding site of the protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15 and mammalian protein L12). An unusual pool of the dephosphorylated forms of proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between the proteins in the pool and on the ribosome takes place during translation. Quadruply disrupted yeast strains, carrying four inactive acidic protein genes and, therefore, containing ribosomes totally depleted of acidic proteins, are viable but grow with a doubling time threefold higher than wild-type cells. The in vitro translation systems derived from these stains are active but the two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in vitro is partially different. These results indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs. Protein PO is analogous to bacterial ribosomal protein L10 but carries an additional carboxyl domain showing a high sequence homology to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD. Successive deletions of the PO carboxyl domain show that removal of the last 21 amino acids from the PO carboxyl domain only slightly affects the ribosome activity in a wild-type genetic background; however, the same deletion is lethal in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional deletions affect the interaction of PO with the P1 and P2 proteins and with the rRNA. The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity.
Asunto(s)
Proteínas Fúngicas/química , Fosfoproteínas/química , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Proteínas Fúngicas/fisiología , Datos de Secuencia Molecular , Fosfoproteínas/fisiología , Fosforilación , Proteínas Ribosómicas/fisiología , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiología , Relación Estructura-ActividadRESUMEN
Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Diferenciación Celular/genética , Sistema Libre de Células , Diploidia , Electroforesis en Gel Bidimensional , Haploidia , Heterocigoto , Mutagénesis , Fosfoproteínas/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/genética , Supresión GenéticaRESUMEN
The sensitivity to different antibiotics of in-vitro polyuridylic acid-dependent polypeptide synthesizing system from Bacteroides vulgatus RYC18F6 and two clindamycin-resistant derivatives was studied. The ribosomes from the resistant strains were not affected by concentrations of up to 0.1 mM clindamycin and lincomycin. In contrast, streptogramin B was found to cause strong stimulation of the clindamycin-resistant polymerizing systems. The modified ribosomes from the resistant strains were more sensitive to other antibiotics like sparsomycin and chloramphenicol. The data indicate that resistance in these B. vulgatus mutant strains is due to alteration of the ribosome structure.