Biology subjects

Aibara, S.

Publications and source records attributed to Aibara, S..

4 recordsLinked to original sources

bioRxiv2021

Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 [A]. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

molecular biology↗

bioRxiv2020

Structure of a transcribing RNA polymerase II-U1 snRNP complex

To initiate co-transcriptional splicing, RNA polymerase II (Pol II) recruits U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent pre-mRNA. Here we report the cryo-EM structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the 5 splice site in pre-mRNA near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5 splice site, leading to formation of an intron loop. Loop formation may facilitate scanning of the nascent pre-mRNA for the 3 splice site and enable prespliceosome assembly and functional pairing of distant intron ends. Our results provide a starting point for a mechanistic analysis of co-transcriptional splicing and the biogenesis of mRNA isoforms during alternative splicing.

molecular biology↗

bioRxiv2020

Assembly and symmetry of the fungal E3BP-containing core of the Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation remain unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components.

molecular biology↗

bioRxiv2020

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit in trypanosomes

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes with reduced RNA and increased protein mass from Trypanosoma brucei, to provide insights into the biogenesis of mitoribosomal large subunit. Structural characterisation of a stable assembly intermediate revealed 22 assembly factors, some of which are also encoded in mammalian genomes. The assembly factors form a protein network that spans over 180 [A], shielding the ribosomal RNA surface. The entire central protuberance and L7/L12 stalk are not assembled, and require removal of the factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly, while the exit tunnel is blocked. Together, the extensive network of the factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

evolutionary biology↗