RNase P is a ubiquitous site-specific endoribonuclease primarily responsible for the maturation of tRNA. Throughout the three domains of life, the canonical form of RNase P is a ribonucleoprotein (RNP) built around a catalytic RNA. The core RNA is well conserved from bacteria to eukaryotes, whereas the protein parts vary significantly. The most complex and the least understood form of RNase P is found in eukaryotes, where multiple essential proteins playing largely unknown roles constitute the bulk of the enzyme. Eukaryotic RNase P was considered intractable to in vitro reconstitution, mostly due to insolubility of its protein components, which hindered its studies. We have developed a robust approach to the in vitro reconstitution of Saccharomyces cerevisiae RNase P RNPs and used it to analyze the interplay and roles of RNase P components. The results eliminate the major obstacle to biochemical and structural studies of eukaryotic RNase P, identify components required for the activation of the catalytic RNA, reveal roles of proteins in the enzyme stability, localize proteins on RNase P RNA, and demonstrate the interdependence of the binding of RNase P protein modules to the core RNA.
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http://dx.doi.org/10.1093/nar/gky333 | DOI Listing |
Methods Mol Biol
January 2025
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, South Korea.
Cell-free in vitro assays offer several advantages for elucidating molecular mechanisms underlying various biological processes. Here, we describe a simple and quantitative in vitro assay using isolated yeast microsomes to measure homotypic ER membrane fusion. In this assay, membrane fusion between ER microsomes is monitored by reconstitution of luciferase activity from split luciferase fragments.
View Article and Find Full Text PDFArch Biochem Biophys
January 2025
Department of Biotechnology, Maharaja Sriram Chandra Bhanjadeo University, (Erstwhile: North Orissa University), Baripada, Odisha, 757003, India. Electronic address:
Our previous study revealed that lipid flip-flop inducing phytochemicals from Gymnema sylvestre increase membrane permeability of antimicrobials in S. aureus. However, their lipid flipping and membrane permeabilizing effect on methicillin resistant S.
View Article and Find Full Text PDFCurr Biol
January 2025
Max Planck Institute for Infection Biology, Virchowweg 12, 10117 Berlin, Germany; Marine Biological Laboratory, 7 Mbl St., Woods Hole, MA 02543, USA; Berliner Hochschule für Technik, Luxemburger Straße 10, 13353 Berlin, Germany. Electronic address:
Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures.
View Article and Find Full Text PDFNat Commun
January 2025
DNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.
Human DNA licensing initiates replication fork assembly and DNA replication. This reaction promotes the loading of the hMCM2-7 complex on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the reconstitution of human DNA licensing using purified proteins.
View Article and Find Full Text PDFZhonghua Yi Xue Yi Chuan Xue Za Zhi
January 2025
Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China.
Objective: To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation.
Methods: A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members.
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