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Showing 1 to 20 of 37 for “"in vitro reconstitution"”.
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In vitro reconstitution of Central Spindle Motility
Precise localisation of intracellular organelles impacts a myriad of cellular processes including cell motility, polarisation and mitosis. Positioning is actively controlled by organelle specific attachment, often involving dedicated motor proteins, to cytoskeletal network. While much is known …
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In vitro reconstitution of the ubiquitylation and disassembly of the eukaryotic replisome
Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the …
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In vitro reconstitution of the human pre-mRNA 3′ end processing machinery and mechanistic insights into endonuclease activation
Maturation of protein-coding transcripts in eukaryotes involves several processing steps, including 5′ capping, splicing, and 3′ end processing. The latter entails endonucleolytic cleavage of the nascent pre-mRNA and addition of a poly(A) tail to the resultant free 3′ end. The poly(A) tail then …
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Investigations of the Mechanism and Substrate Scope of the Enzymes Involved in Lantibiotic Biosynthesis
The subtilin prepeptide SpaS consists of 56 amino acids that includes five cysteine residues. This peptide is the substrate of the putative lantibiotic dehydratase, SpaB, and cyclase SpaC. The heterologously expressed and purified SpaS peptide was employed in assays with the purified SpaB and SpaC …
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From Peptide Precursors to Lanthionine -Containing Peptide Antibiotics Using the First Isolated and Active Lacticin 481 Synthetase: Mechanistic Studies and Peptide Engineering
The putative dehydratase SpaB involved in the biosynthesis of subtilin was heterologously overexpressed in E. coli and purified by metal-chelated affinity chromatography. However, in vitro reconstitution using the purified SpaB, SpaC, and the substrate SpaS was unsuccessful. As a result, the …
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Post-translational modifications catalyzed by RiPP and RiPP-adjacent enzymes
… utilized by Nature to transform these peptides into structurally fascinating natural products is astounding. Leveraging these enzymes for novel uses has and will continue to provide value. However, before they can be engineered, we much first understand how these enzymes function, what their …
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Mechanistic Studies of Lantibiotic Biosynthetic Enzymes and Discovery of Novel Lanthionine-Containing Peptides
… synthesized antimicrobial peptides containing unique thioether crosslinks, lanthionines and methyllanthionines. These motifs are introduced posttranslationally in a two-step process, which involves the dehydration of serines and threonines into dehydroalanines and dehydrobutyrines, …
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Exploring Prokaryotic Thiamin Biosynthesis: Mechanistic Studies On Thiamin Thiazole Synthase And Pyrimidine Synthase
Thiamin (Vitamin B1) is made from a coupling a thiazole and a pyrimidine unit, which are assembled separately. Studies have shown that the biosyntheses of thiazole and pyrimidine are different in prokaryotes versus eukaryotes. Understanding of thiamin biosynthesis is still incomplete and a lot of …
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ARCHITECTURE AND RNA RECOGNITION BY THE EUKARYOTIC M6A METHYLTRANSFERASE COMPLEX
… regulators of gene expression, influencing diverse biological functions. Among them, N6-methyladenosine (m6A) is one of the most abundant mRNA modifications and contributes to key cellular processes, including mRNA processing, translation, and decay. In eukaryotes, m6A is …
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Regulation of polarised trafficking by Klp98A
Directional trafficking of cargo, a hallmark of living cells, relies on a polarised landscape of microtubule tracks. Here, I studied how the regulation of an endosomal motor affects polarised trafficking, using the asymmetric division of *Drosophila* Sensory Organ Precursor (SOP) cells as a model …
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Regulation of mammalian IRE1α: Co-chaperones and their importance
When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of …
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Spatio-temporal properties of membrane-localized actin nucleating complexes
The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that …
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Structural and functional effects of histone variant, H2A.Bbd, on the nucleosome core particle
In eukaryotic cells, DNA is packaged into a protein-DNA assembly called chromatin. The basic subunit of chromatin is the nucleosome core, which is composed of 147 base pair (bp) of DNA wrapped in 1.65 turns around a histone octamer containing two copies each of the four core histone proteins (H2A, …
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Mechanistic Analysis of Microtubule Dynamics and Regulation
Microtubules are critical components in a cells cytoskeletal network, known to form the mitotic spindle which allows for chromosome segregation and cell division and organizing the cytoplasm of non-dividing cells. The quick reorganization of the cytoskeleton relies heavily on the underlying …
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A Platform for Analyzing Force Sensitivity and Multivalency in Actin Networks
… of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them …
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MOLECULAR MECHANISMS OF THE FORMATION OF ENDOCYTIC PATCHES AND THE INITIATION OF ACTIN POLYMERIZATION IN FAST ENDOPHILIN MEDIATED ENDOCYTOSIS
Endocytosis is an essential cellular process that involves the internalization of a varietyof cargo including macronutrients and transmembrane receptors. Fast endophilin mediated endocytosis (FEME) is a clathrin independent endocytic pathway that is responsible for the uptake of numerous receptors, …
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Mechanistic analysis of the Zika virus translation-replication switch
… required for both translation and replication during infection. These two processes are antagonistic in nature, each requiring the RNA template in opposite directions. Thus, the balance between these processes during infection must be tightly regulated. Zika virus (ZIKV) is a capped, …
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Biochemical and Structural Studies of RNA and DNA Editing Enzymes
RNA and DNA editing, occurring by way of hydrolytic deamination of cytidine (C) and adenosine (A), is a modification event that profoundly effects on an organisms vitality and quality of life. This study focuses on two types of enzymes---ADAT (adenosine deaminase acting on tRNA) and APOBEC3G …
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The eIF2 phosphatase: characterization and modulation
Cellular needs are fulfilled by the combined activity of functional proteins. Consequently, cells are equipped with a complex proteostatic network that controls protein production in response to cellular requisites. Thereby, protein synthesis is induced or attenuated depending on the particular …
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A genomics-led approach to deciphering heterocyclic natural product biosynthesis
Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the …
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