A5066955881- RNA and protein synthesis mechanisms
- Genomics and Chromatin Dynamics
- RNA Research and Splicing
- RNA modifications and cancer
- Fungal and yeast genetics research
- Bacterial Genetics and Biotechnology
- Advanced Electron Microscopy Techniques and Applications
- Calcium signaling and nucleotide metabolism
- Gout, Hyperuricemia, Uric Acid
- Inflammasome and immune disorders
- Heat shock proteins research
- Genetic and Kidney Cyst Diseases
- DNA Repair Mechanisms
- CRISPR and Genetic Engineering
- Molecular Biology Techniques and Applications
- Electron and X-Ray Spectroscopy Techniques
- Cancer-related molecular mechanisms research
- Cancer Genomics and Diagnostics
- Biomedical Research and Pathophysiology
- Forensic and Genetic Research
- Microtubule and mitosis dynamics
University of Regensburg
2014-2024
Abstract Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level production relies on efficient binding initiation factors the promoter and recruitment Pol complexes containing factor Rrn3. Here, we determine cryo-EM structure I-Rrn3 complex at 7.5 Å resolution, compare it with Rrn3-free monomeric dimeric I. We observe that Rrn3 contacts A43/A14 stalk subunits A190 AC40, association re-organizes interaction interface,...
In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol a determinant cellular growth lifespan across eukaryotes. Upregulation observed in cancer causative mutations have been described neurodevelopmental disorders hypersensitivity to viral infection. Here, we report cryo-EM reconstruction at 4.0 Å human III, allowing mapping rationalization reported genetic mutations. Mutations causing defects cluster hotspots affecting...
Abstract Transcription of the ribosomal RNA precursor by polymerase (Pol) I is a prerequisite for biosynthesis ribosomes in eukaryotes. Compared to Pols II and III, mechanisms underlying promoter recognition, initiation complex formation DNA melting Pol substantially diverge. Here, we report high-resolution cryo-EM reconstruction early intermediate assembled on double-stranded scaffold that prevents establishment downstream contacts. Our analyses demonstrate how efficient promoter-backbone...
Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination rDNA by RNA polymerase I (Pol I) the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like binding domains and associates vivo near 3′ end rRNA genes rDNA, but information about which sequences might influence Nsi1-dependent lacking. Here, we show that stretch 11 nucleotides...
Abstract Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal factors, only Spt5 found all domains life, but has been shaped during evolution, which also reflected heterodimerization with Spt4 Archaea Eukaryotes. To unravel mechanistic basis Spt4/5 Archaea, we performed structure-function analyses using archaeal transcriptional machinery Pyrococcus furiosus (Pfu). We...
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal (rRNA) precursor. In budding yeast, Pol multimeric enzyme with 14 subunits. Among them, Rpa49 forms Rpa34 I-specific heterodimer (homologous to PAF53/CAST in human might be responsible for specific functions I. Previous studies provided insight involvement initiation, elongation, docking and releasing Rrn3, an essential transcription factor. Here, we took...
RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol transcription machinery has to deal with nucleosomal barriers. It been suggested that I-associated factors facilitate chromatin transcription, but it unknown whether an intrinsic capacity transcribe through nucleosomes. Here, we used vitro assays study purified WT and mutant variants from yeast Saccharomyces cerevisiae...
Transcription of the ribosomal RNA precursor by polymerase (Pol) I is a major determinant cellular growth, and dysregulation observed in many cancer types. Here, we present purification human Pol from cells carrying genomic GFP fusion on largest subunit allowing structural functional analysis enzyme across species. In contrast to yeast, carries single-subunit stalk, vitro transcription indicates reduced proofreading activity. Determination cryo-EM reconstruction close-to-native state...
RNA polymerase I (Pol I) synthesizes ribosomal (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity proliferating cells. Although basal Pol transcription factors have been characterized diverse organisms, molecular basis robust rRNA production vivo remains largely unknown. In S. cerevisiae, multifunctional Net1 protein was reported to stimulate transcription. We found that I-stimulating function can be attributed very C-terminal region (CTR) Net1. The CTR...
Abstract NLRP3 is an intracellular sensor protein whose activation by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation pyroptosis. The mechanisms leading the way how antagonistic small molecules function remain poorly understood. Here we report cryo-electron microscopy structures full-length in its native form complexed with inhibitor CRID3 (also named MCC950). Inactive, ADP-bound decamer composed homodimers intertwined LRR domains that assemble...
Abstract Recent technological progress revealed new prospects of high-resolution structure determination macromolecular complexes using cryo-electron microscopy (cryo-EM). In the field RNA polymerase (Pol) I research, a number cryo-EM studies contributed to understanding highly specialized mechanisms underlying transcription ribosomal genes. Despite broad applicability method itself, preparation samples for data collection can be challenging. Here, we describe strategies purification and...
In yeast and human cells many of the ribosomal proteins (r-proteins) are required for stabilisation productive processing rRNA precursors. Functional coupling r-protein assembly with maturation subunit precursors potentially promotes production ribosomes defined composition. To further decipher mechanisms such an intrinsic quality control pathway we analysed here contribution three large r-proteins rpL2 (uL2), rpL25 (uL23) rpL34 (eL34) intermediate nuclear folding steps. Structure models...
Abstract Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal factors, only Spt5 found all domains life but has been shaped during evolution, which also reflected heterodimerization with Spt4 Archaea Eukaryotes. To unravel mechanistic basis Spt4/5 Archaea, we performed structure-function analyses using archaeal transcriptional machinery Pyrococcus furiosus ( Pfu ). We...
Abstract Molecular machines play pivotal roles in all biological processes. Most structural methods, however, are unable to directly probe molecular motions. Here, we demonstrate that dedicated NMR experiments can provide quantitative insights into functionally important dynamic regions very large asymmetric protein complexes. We establish this for the 410 kDa eukaryotic RNA exosome complex contains ten distinct chains. Methyl-group and fluorine reveal site-specific interactions among...
Abstract Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal (rRNA) precursor. In budding yeast, Pol multimeric enzyme with 14 subunits. Among them, Rpa49 forms Rpa34 I-specific heterodimer (homologous to PAF53/CAST in human might be responsible for specific functions I. Previous studies provided insight involvement initiation, elongation, docking and releasing Rrn3, an essential transcription factor. Here,...
Abstract Transcription of the ribosomal RNA precursor by polymerase (Pol) I is a major determinant cellular growth and dysregulation observed in many cancer types. Here, we present purification human Pol from cells carrying genomic GFP-fusion on largest subunit allowing structural functional analysis enzyme across species. In contrast to yeast, carries single-subunit stalk vitro transcription indicates reduced proofreading activity. Determination cryo-EM reconstruction close-to-native state...
Abstract Eukaryotic RNA polymerases I and III (Pol III) consist of core subunits, which are conserved in polymerase II II). Additionally, Pol have specific associating with the so-called ‘lobe’ structure first described within II. In yeast S. cerevisiae , these Rpa34.5, N-terminal domains Rpa49 Rpa12.2, here referred to as lobe-binding module (lb-module). We analyzed functions lb-module a defined vitro transcription system. Cooperation between components influenced fidelity, elongation...