RESUMEN
The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.
Asunto(s)
Microscopía por Crioelectrón , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/ultraestructura , Origen de Réplica , Saccharomyces cerevisiae , Secuencia de Bases , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Moleculares , Complejo de Reconocimiento del Origen/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Especificidad por SustratoRESUMEN
The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Proteínas de Mantenimiento de Minicromosoma/química , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adenilil Imidodifosfato/química , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Microscopía por Crioelectrón , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/ultraestructura , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Dedos de ZincRESUMEN
We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.
Asunto(s)
Cromosomas Artificiales de Levadura/química , ADN Ribosómico/genética , Ingeniería Genética/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Cromosomas Artificiales de Levadura/genética , Cromosomas Artificiales de Levadura/ultraestructura , Saccharomyces cerevisiae/ultraestructura , TranscriptomaRESUMEN
EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.