分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 angstrom, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: DNA methylation is an important epigenetic modification that is essential for various developmental processes through regulating gene expression, genomic imprinting, and epigenetic inheritance(1-5). Mammalian genomic DNA methylation is established during embryogenesis by de novo DNA methyltransferases, DNMT3A and DNMT3B(6-8), and the methylation patterns vary with developmental stages and cell types(9-12). DNAmethyltransferase 3-like protein (DNMT3L) is a catalytically inactive paralogue of DNMT3 enzymes, which stimulates the enzymatic activity of Dnmt3a(13). Recent studies have established a connection between DNA methylation and histone modifications, and revealed a histone-guided mechanism for the establishment of DNA methylation(14). The ATRX-DNMT3-DNMT3L (ADD) domain of Dnmt3a recognizes unmethylated histone H3 (H3K4me0)(15-17). The histone H3 tail stimulates the enzymatic activity of Dnmt3a in vitro(17,18), whereas the molecular mechanism remains elusive. Here we show that DNMT3A exists in an autoinhibitory form and that the histone H3 tail stimulates its activity in a DNMT3L-independent manner. We determine the crystal structures of DNMT3A-DNMT3L (autoinhibitory form) and DNMT3A-DNMT3L-H3 (active form) complexes at 3.82 and 2.90 angstrom resolution, respectively. Structural and biochemical analyses indicate that the ADD domain of DNMT3A interacts with and inhibits enzymatic activity of the catalyticdomain (CD) through blocking its DNA-binding affinity. HistoneH3(but not H3K4me3) disrupts ADD-CD interaction, induces a large movement of the ADD domain, and thus releases the autoinhibition of DNMT3A. The finding adds another layer of regulation of DNA methylation to ensure that the enzyme is mainly activated at proper targeting loci when unmethylated H3K4 is present, and strongly supports a negative correlation between H3K4me3 and DNA methylation across the mammalian genome(9,10,19,20). Our study provides a new insight into an unexpected autoinhibition and histone H3-induced activation of the de novo DNA methyltransferase after its initial genomic positioning.