A5060049543- RNA and protein synthesis mechanisms
- RNA Research and Splicing
- Genomics and Chromatin Dynamics
- RNA modifications and cancer
- DNA and Nucleic Acid Chemistry
- Diatoms and Algae Research
- Photoreceptor and optogenetics research
- Bacterial Genetics and Biotechnology
- DNA Repair Mechanisms
- Advanced biosensing and bioanalysis techniques
- CRISPR and Genetic Engineering
- Chromosomal and Genetic Variations
- Neural dynamics and brain function
- Cancer therapeutics and mechanisms
- Mosquito-borne diseases and control
- Advanced Electron Microscopy Techniques and Applications
- Enzyme Structure and Function
- Neuroscience and Neuropharmacology Research
- Plant Genetic and Mutation Studies
- Biofuel production and bioconversion
- Systemic Lupus Erythematosus Research
- Marine Sponges and Natural Products
- Advances in Cucurbitaceae Research
- Energy and Environment Impacts
- Diet and metabolism studies
RIKEN Center for Biosystems Dynamics Research
2018-2025
RIKEN
2024
Systems Biology Institute
2011-2017
The University of Tokyo
2011-2017
Kyowa Kirin (Japan)
2010
Transcription machinery remains steadfast Eukaryotic transcription of mRNA is a multistep process mediated by RNA polymerase II (Pol II). Pol combines with several other factors to form an elongation complex that promotes elongation. Ehara et al. determined the high-resolution structure means x-ray crystallography and cryo-electron microscopy (see Perspective Fouqueau Werner). Multiple are distributed on wide surface establish exit path DNA entry or tunnels, which facilitate nascent...
Genomic DNA forms chromatin, in which the nucleosome is repeating unit. The mechanism by RNA polymerase II (RNAPII) transcribes nucleosomal remains unclear. Here we report cryo-electron microscopy structures of RNAPII-nucleosome complexes RNAPII pauses at superhelical locations SHL(-6), SHL(-5), SHL(-2), and SHL(-1) nucleosome. major histone-DNA contact sites, interactions with subunits stabilize pause. These reveal snapshots transcription, gradually tears from histone surface while...
RNA polymerase II (RNAPII) transcribes chromosomal DNA that contains multiple nucleosomes. The nucleosome forms transcriptional barriers, and nucleosomal transcription requires several additional factors in vivo. We demonstrate the elongation Elf1 Spt4/5 cooperatively lower barriers increase RNAPII processivity nucleosome. cryo-electron microscopy structures of nucleosome-transcribing complexes (ECs) reveal reshape EC downstream edge intervene between They facilitate progression through...
During gene transcription, RNA polymerase II (RNAPII) traverses nucleosomes in chromatin, but the mechanism has remained elusive. Using cryo–electron microscopy, we obtained structures of RNAPII elongation complex (EC) passing through a nucleosome presence transcription factors Spt6, Spn1, Elf1, Spt4/5, and Paf1C histone chaperone FACT (facilitates chromatin transcription). The show snapshots EC progression on DNA mediating downstream disassembly, followed by its reassembly upstream EC,...
Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), member of the flaviviruses. The DENV genome 5'-capped positive-sense RNA with unique 5'-stem-loop structure (SLA), which essential for replication and 5' capping. virus-encoded proteins NS5 NS3 are responsible replication, but structural basis they cooperatively conduct required tasks has remained unclear. Here, we report cryoelectron microscopy (cryo-EM) structures SLA-bound (PC), NS3-bound PC (PC-NS3), an...
Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, mediated the adenosine triphosphate-dependent translocase/helicase Rho, which causes RNA/DNA dissociation from RNAP elongation complex (EC). However, structural basis of interplay between Rho remains obscure. Here, we report cryo-electron microscopy structure Thermus thermophilus EC engaged with Rho. The hexamer binds through carboxyl-terminal...
The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that nucleosome containing H3E97K extremely unstable as compared to wild-type nucleosome. However, mechanism by which causes instability has not been clarified yet. In present study, cryo-electron microscopy structure entry/exit DNA regions are disordered, probably detachment nucleosomal from H3 N-terminal regions. This may change intra-molecular amino acid...
The metazoan transcription elongation complex (EC) of RNA polymerase II (RNAPII) generally stalls between the start site and first (+1) nucleosome. This promoter-proximal pausing involves negative factor (NELF), 5,6-dichloro-1-β-d-ribobenzimidazole sensitivity-inducing (DSIF), IIS (TFIIS) is critical for subsequent productive elongation. However, detailed mechanism involvement +1 nucleosome remain enigmatic. Here, we report cryo-electron microscopy structures ECs stalled on nucleosomal DNA....
Vα14 TCR expressing invariant NK T (iNKT) cells recognize α-galactosylceramide (αGC)/CD1d complex and produce large amounts of various cytokines before the onset adaptive immunity. After stimulation with a high dose (2-5 μg) αGC in vivo, iNKT spleen liver become anergic terms proliferation cytokine production to subsequent stimulation. In this study, we monitor how anergy is induced. Anergized dramatically reduced expression IL-2Rα, exogenous IL-2 restored ability proliferate IL-4 but not...
Rac1-bound structure and mutagenesis of the ELMO1-DOCK5 complex provide insights into how ELMO modulates DOCK activity.
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, mechanism by which conformational pump an anion to achieve unidirectional transport, from extracellular side cytoplasmic side, anion-pumping remains enigmatic. We collected...
Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes to introduce the H3 lysine 27 tri-methyl (H3K27me3) chromatin mark. Although this mechanism provides a link between two key transcriptional repressors, PRC2, it is unknown how histone-tail dynamics contribute process. Here, we have examined effect of ubiquitination linker-DNA on H3-tail H3K27 methylation by PRC2. In naïve...
The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation methylation. Histone is considered to enhance transcription in chromatin. However, contribution tail by RNA polymerase II (RNAPII) has not been clarified. In present study, we reconstituted nucleosomes lacking each histone, H2A, H2B, H3 or H4, performed RNAPII assays. We found that H3, but H2B functions pausing at SHL(-5) position nucleosome. Consistently,...
Abstract The 5’-3’ exoribonuclease Rat1 (or Xrn2) plays a pivotal role in termination of mRNA transcription by eukaryotic RNA polymerase II (RNAPII). forms complex with its partner proteins, Rai1 and Rtt103, acts as “torpedo” to bind transcribing RNAPII dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures Rat1-Rai1-Rtt103 three Rat1-Rai1-associated complexes (type-1, type-1b, type-2) yeast Komagataella phaffii . structure revealed that form heterotetramer,...
The function of the mitogen-activated protein kinase signaling pathway is required for activation immediate early genes (IEGs), including EGR1 and FOS, cell growth proliferation. Recent studies have identified topoisomerase II (TOP2) as one important regulators transcriptional IEGs. However, mechanism underlying regulation involving TOP2 in IEG has remained unknown. Here, we demonstrate that ERK2, but not ERK1, report a critical ELK1 binding sequence ERK2 at gene. Our data indicate both ERK1...
Komagataella pastoris is a methylotrophic yeast that commonly used as host cell for protein production. In the present study, we reconstituted nucleosome with K. histones and determined structure of core particle by cryogenic electron microscopy. nucleosome, form an octamer DNA left-handedly wrapped around it. Micrococcal nuclease assays revealed ends are somewhat more accessible, compared those human nucleosome. vitro transcription demonstrated transcribed RNA polymerase II (RNAPII)...
Abstract In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, mechanism by which RNA polymerase II (RNAPII) transcribes DNA in chromatosome has remained enigmatic. Here we report cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I II), RNAPII is paused at entry region due binding. form complex, bound nucleosome restricts orientation, exit captured binding cleft. progresses...
RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) SHL(-1), respectively]. In present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused a major pausing site, SHL(-1). We determined two previously undetected structures, which transcribed behind RNAPII is sharply kinked exit tunnel rewrapped around histones front looping. This kink shifts...
RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, crystal structure Pol from methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While highly homologous to budding Saccharomyces cerevisiae, stalk clamp modules K. displayed large inward rotations, closing central cleft greater extent than in known S. cerevisiae structures. The conformational differences reflect inherent flexibilities...
We developed a method for efficient chromosome tagging in Pichia pastoris, using useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF contains thrombin protease cleavage site removal of tag hexahistidine sequence (6× His) followed by three copies FLAG (3× FLAG) purification. Using this method, THF-tagged RNA polymerases I, II, III were successfully purified from P. pastoris. also enabled us to purify tagged polymerase II on large scale, its...