RESUMEN
The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.
Asunto(s)
Chaperonina con TCP-1/genética , Mutación , Actinas , Adaptación Fisiológica/genética , Proteínas de Ciclo Celular , Chaperonina con TCP-1/fisiología , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Fosfatidilinositol 3-Quinasas , Subunidades de Proteína/genética , Ribosomas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
RNA protection experiments and the crystal structure of a complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with rapamycin, a polyketide compound resembling macrolides and ketolides, showed that rapamycin binds to a crevice located at the boundaries of the nascent protein exit tunnel, near its entrance. At this location rapamycin cannot occlude the ribosome exit tunnel, consistent with its failure to act as a ribosomal antibiotic drug. In accord with recent biochemical data, this crevice may play a role in facilitating local cotranslational folding of nascent chains, in particular for transmembrane proteins.
Asunto(s)
Deinococcus/química , Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/química , Ribosomas/metabolismo , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , Ribosomas/efectos de los fármacos , Sirolimus/química , Sirolimus/metabolismo , Sirolimus/farmacologíaRESUMEN
The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.
