RESUMO
Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.
Assuntos
Cloreto de Lítio/toxicidade , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Marcação por Isótopo , Cloreto de Lítio/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Estrutura Quaternária de Proteína , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.
