RESUMO
Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane environment. The development of the pBEVY vector system has further increased the potential of S. cerevisiae as an expression system by creating a method for expressing multiple proteins simultaneously. This vector system is amenable to the expression and purification of multi-subunit protein complexes. Here we describe the cloning, yeast transformation, and co-expression of multi-subunit outer mitochondrial membrane complexes using the pBEVY vector system.
Assuntos
Clonagem Molecular/métodos , Proteínas de Membrana , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fracionamento Celular/métodos , Regulação Fúngica da Expressão Gênica , Vetores Genéticos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Organismos Geneticamente Modificados , Multimerização Proteica/genética , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process.
Assuntos
Mitocôndrias/genética , RNA de Protozoário/química , RNA/química , Uridina/química , Quadruplex G , Kinetoplastida/química , Kinetoplastida/genética , Mitocôndrias/química , RNA/genética , Edição de RNA , Motivo de Reconhecimento de RNA/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Uridina/genéticaRESUMO
Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life.
Assuntos
Proteínas Arqueais/química , Centrômero/química , Segregação de Cromossomos , Cromossomos de Archaea/genética , DNA Arqueal/genética , Sulfolobus/genética , Motivos de Aminoácidos , Proteínas Arqueais/genética , Autoantígenos/química , Autoantígenos/genética , Bactérias/genética , Centrômero/genética , Proteína Centromérica A , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , DNA Arqueal/química , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Kluyveromyces/genética , Conformação de Ácido Nucleico , Estrutura Terciária de ProteínaRESUMO
Kinetoplastid RNA (kRNA) editing is a process that creates translatable mitochondrial mRNA transcripts from cryptogene encoded RNAs and is unique for kinetoplastids, such as Trypanosoma brucei. In addition to the catalytic 20S editosome, multiple accessory proteins are required for this conversion. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. MRB1 consists of six core proteins but makes dynamic interactions with additional accessory proteins. Here we describe the characterization of one such factor, the 72 kDa MRB1590 protein. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). Structural studies show that MRB1590 is dimeric and contains a central ABC-ATPase fold embedded between novel N- and C-terminal regions. The N-terminal domains combine to create a basic pore and biochemical studies indicate residues in this region participate in RNA binding. Structures capturing distinct MRB1590 conformations reveal that the RNA binding pore adopts closed and open states, with the latter able to accommodate RNA. Based on these findings, implications for MRB1590 function are discussed.
