Biology subjects

Schmitzova, J.

Publications and source records attributed to Schmitzova, J..

6 recordsLinked to original sources

bioRxiv2025

Structure of human cytoplasmic Pol II complex explains global transcription repression by Gdown1

RNA polymerase II (Pol II) is a 12-subunit enzyme crucial for gene transcription in the nucleus. However, its assembly in the cytoplasm, nuclear import, and nuclear function of assembly factors remain poorly understood. Here, we isolated Pol II from the cytoplasmic fraction of human cells (cfPol II) and determined its cryo-EM structure. The structure reveals that Pol II is fully assembled in the cytoplasm before nuclear import. We also found that Gdown1 binds Pol II through three distinct regions, indicating it may stabilize Pol II assembly intermediates. Notably, Gdown1 binding precludes the association of essential transcription factors IIB and IIF, rendering cfPol II inactive in promoter-dependent transcription in vitro. Our results provide a basis for Gdown1-dependent global transcription repression and suggest a model for the role of Gdown1 in Pol II assembly, import, and transcription regulation.

biochemistry↗

bioRxiv2024

Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

biochemistry↗

bioRxiv2021

An integrated model for termination of RNA polymerase III transcription

RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

genomics↗

bioRxiv2021

Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 patients1,2. Molnupiravir increases the frequency of viral RNA mutations3,4 and impairs SARS-CoV-2 replication in animal models4-6 and in patients2. Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase (RdRp) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp readily uses the active form of molnupiravir, {beta}-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp progression. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

biochemistry↗

bioRxiv2021

Dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 [A] resolution. In this structure, the two RdRps contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs for transcription.

molecular biology↗

bioRxiv2020

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1-4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25-7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

biochemistry↗