Current research

Transcription in chromatin
In eukaryotes, genomic DNA is packaged in the cell nucleus as chromatin, which consists of multiple basic units called nucleosomes. RNA polymerase II (RNAPII) is the enzyme responsible for gene transcription. It transcribes DNA sequences, while translocating across chromatinized DNA within the gene. Remarkably, RNAPII is able to carry out transcription without disrupting chromatin structure, rather maintaining its integrity throughout this process. This sophisticated mechanism is achieved by a variety of factors that associate with transcribing RNAPII and nucleosomes. By reconstituting RNAPII–nucleosome complexes engaged in transcription and analyzing their structures using cryo-electron microscopy (cryo-EM), we have elucidated the mechanisms of transcription within chromatin. We revealed that an elongation complex of RNAPII traverses a nucleosome by temporarily disassembling the nucleosome, followed by reassembling it behind the complex. Furthermore, we uncovered the mechanism by which epigenetic histone modifications are introduced during transcription, in a transcription-coupled manner.

Promoter-proximal regulation
In metazoans, RNAPII that has initiated transcription from a gene promoter generally pauses within a region 20–60 base pairs downstream of the transcription start site. This phenomenon is known as promoter-proximal pausing, which plays essentail roles in proper RNA processing and embryonic development. In addition to negative elongation factors, NELF and DSIF, the first nucleosome immediately downstream of the transcription start site (the +1 nucleosome) is known to be involved in the pausing. By reconstituting RNAPII complexes paused on nucleosomal DNA and determining their structures using cryo-EM, we have elucidated the mechanism by which the negative elongation factors and the +1 nucleosome cooperativly establish the promoter-proximal checkpoint.

Transcription termination mechanisms
At the final stage of transcription, termination factors are recruited to transcribing RNAPII near the termination site, leading to transcription termination (dissociation of RNAPII from DNA). The yeast transcription termination factor Rat1 is an RNA exonuclease, which reportedly induces termination by degrading RNA and colliding with the RNAPII as a “torpedo”. By reconstituting and determining the structure of a pre-termination complex, we elucidated this torpedo mechanism. Rat1 binds near the RNA exit site of RNAPII and captures the 5' end of the RNA into its active site, suggesting that triggers termination by destabilizing the transcription complex. We also determined the structure of a bacterial pre-termination complex, in which the bacterial termination factor Rho, an ATP-dependent RNA helicase, is bound to RNAP, revealing similarities between transcription termination mechanisms in eukaryotes and bacteria.

Viral replication mechanisms
Dengue virus, a single-stranded RNA virus, is the causative agent of dengue, a mosquito-borne infection. Approximately half of the world’s population is at risk of dengue virus infection, but effective therapeutic agents are currently unavailable. Within infected human cells, dengue virus replicates its RNA genome using viral-encoded enzymes, NS5 and NS3. We have successfully determined the structures of multiple complexes formed between NS5, NS3 and RNA, thereby shedding new light on the mechanisms of the dengue virus RNA replication. These structural insights would provide a foundation for future structure-based drug design.