RESUMO
Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.
Assuntos
Clonagem Molecular/métodos , Complexos Multiproteicos/química , Conformação Proteica , Saccharomyces cerevisiae , Sequência de Aminoácidos , Calorimetria/métodos , Cristalografia por Raios X/métodos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Espalhamento a Baixo Ângulo , Spliceossomos/química , Difração de Raios X/métodosAssuntos
Endorribonucleases/metabolismo , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Mutação , Fenótipo , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/isolamento & purificação , RNA Helicases/genética , RNA Helicases/isolamento & purificação , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismoRESUMO
The yeast nuclear gene DSS1 codes for a mitochondrial protein containing regions of homology to bacterial RNase II and can act as a multicopy suppressor of a deletion of the SUV3 gene, which encodes an RNA helicase. In order to establish the function of the DSS1 gene in mitochondrial biogenesis we studied RNA metabolism in yeast strains disrupted for SUV3 or DSS1. The results indicate that in the absence of DSS1 the in vitro activity of 3'-5' exoribonuclease is abolished and mitochondrial translation is blocked. In disruption strains harboring intronless mitochondrial genomes steady-state levels of COB mRNA and 16S rRNA were very low, while in the presence of a mitochondrial genome containing the omega intron in the 21S rRNA gene the excised intron accumulates. Moreover we observed an accumulation of precursors of 21S rRNA and the VAR1 mRNA. All these phenotypes are virtually identical to those of strains in which SUV3 is disrupted. We suggest that the DSS1 gene product, like the SUV3 gene product, is a subunit of the yeast mitochondrial degradosome (mtEXO), and that this protein complex participates in intron-independent turnover and processing of mitochondrial transcripts. In addition our studies exclude any role for the NUC1 nuclease in these phenomena.
Assuntos
Endorribonucleases/metabolismo , Exorribonucleases/genética , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animais , Núcleo Celular/genética , Cricetinae , RNA Helicases DEAD-box , Proteínas Fúngicas/genética , Genes Fúngicos , RNA Helicases/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/enzimologiaRESUMO
Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.
