Biology subjects

Skubnik, K.

Publications and source records attributed to Skubnik, K..

5 recordsLinked to original sources

bioRxiv2025

Mechanisms of transcription attenuation and condensation of RNA polymerase II by RECQ5

The elongation rates of RNA polymerase II (RNAPII) require precise control to prevent transcriptional stress, which can impede co-transcriptional pre-mRNA processing and contribute to many age- or disease-associated molecular changes (e.g., loss of proteostasis)1-5. Additionally mesoscale organization of transcription is thought to control the transcriptional rates6 and multiple factors have been reported to form biomolecular condensates and integrate RNAPII through the interaction with the C-terminal domain (CTD) of the largest subunit, RPB17-10. However, the structural organization of these condensates remains uncharacterized due to their small size and inherently dynamic nature. Here, we investigated the molecular mechanisms by which a general transcription factor - RECQ5 - associates with hyperphosphorylated RNAPII elongation complex (P-RNAPII EC) and controls translocation of RNAPII along genes. We combined biochemical reconstitution, electron cryomicroscopy, cryotomography, and coarse-grained simulations. We report two mechanisms by which RECQ5 modulates RNAPII transcription. At the atomic level, we demonstrate that RECQ5 uses the brake helix as a doorstop to control RNAPII translocation along DNA, attenuating transcription. At the mesoscale level, RECQ5 forms a condensate scaffold matrix, integrating P-RNAPII EC through a network of site-specific interactions, reinforcing the condensates structural integrity. Our integrative, multi-scale study provides insights into the structural basis of transcription attenuation and into the molecular architecture and biogenesis of a model RNAPII condensate.

biochemistry↗

bioRxiv2024

Cell attachment and tail contraction of S. aureus phage phi812

Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.

microbiology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗

bioRxiv2022

Human myelin proteolipid protein structure and lipid bilayer stacking

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, -helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

biochemistry↗

bioRxiv2018

Structure and genome ejection mechanism of Podoviridae phage P68 infecting Staphylococcus aureus

Phages infecting S. aureus have the potential to be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here we present the structures of S. aureus phage P68 in its native form, genome ejection intermediate, and empty particle. The P68 head contains seventy-two subunits of inner core protein, fifteen of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. P68 genome ejection is triggered by disruption of the interaction of one of the portal protein subunits with phage DNA. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.

microbiology↗