1,721,066 research outputs found
Rewiring translation – Genetic code expansion and its applications
No full textAbstractWith few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNACUA pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies
In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding
No full textHistone chaperones assist nucleosomal rearrangements to facilitate the passage of DNA and RNA polymerases through chromatin. The FACT (facilitates chromatin transcription) complex is a conserved histone chaperone involved in transcription, replication, and repair. The complex consists of two major subunits, Spt16 and SSRP1/Pob3 in mammals and yeast, which engage histones and DNA by multiple contacts. However, the precise mechanism of FACT function is largely unclear. Here, we used the genetically installed UV-activatable cross-linker amino acid p-benzoylphenylalanine (pBPA) to map the interaction network of FACT in living yeast. Unexpectedly, we found the acidic C-terminus of Pob3 forming cross-links to histone H2A and H2B most efficiently. This observation was independent of the performed cross-linking chemistry since similar histone cross-links were obtained using p-azidophenylalanine (pAzF). Further analyses identified a C-terminal nuclear localization sequence in Pob3. Its interaction with Importin-alpha interfered with H2A H2B binding, which suggests a possible regulatory role in FACT recruitment to chromatin. Deletion of acidic residues from the Pob3 C-terminus creates a hydroxyurea-sensitive phenotype in budding yeast, suggesting a potential role for this domain in DNA replication
Chromosome condensation and decondensation during mitosis
No full textDuring eukaryotic cell division, nuclear chromatin undergoes marked changes with respect to shape and degree of compaction. Although already significantly compacted during interphase, upon entry into mitosis chromatin further condenses and individualizes to discrete chromosomes that are captured and moved independently by the mitotic spindle apparatus. Once segregated by the spindle, chromatin decondenses to re-establish its interphase structure competent for DNA replication and transcription. Although cytologically described a long time ago, the underlying molecular mechanisms of mitotic chromatin condensation and decondensation are still ill-defined. Here we summarize our current knowledge of mitotic chromatin restructuring and recent progress in the field
Synthetic biology approaches in drug discovery and pharmaceutical biotechnology
Full text linkSynthetic biology is the attempt to apply the concepts of engineering to biological systems with the aim to create organisms with new emergent properties. These organisms might have desirable novel biosynthetic capabilities, act as biosensors or help us to understand the intricacies of living systems. This approach has the potential to assist the discovery and production of pharmaceutical compounds at various stages. New sources of bioactive compounds can be created in the form of genetically encoded small molecule libraries. The recombination of individual parts has been employed to design proteins that act as biosensors, which could be used to identify and quantify molecules of interest. New biosynthetic pathways may be designed by stitching together enzymes with desired activities, and genetic code expansion can be used to introduce new functionalities into peptides and proteins to increase their chemical scope and biological stability. This review aims to give an insight into recently developed individual components and modules that might serve as parts in a synthetic biology approach to pharmaceutical biotechnology.German Initiative of Excellence; German Research Foundatio
The use of unnatural amino acids to study and engineer protein function
No full textThe expansion of the genetic code for the incorporation of unnatural amino acids (UAAs) in proteins of bacteria, yeasts, mammalian cells or whole animals provides molecular and structural biologists with an amazing kit of novel tools. UAAs can be used to investigate the structure and dynamics of proteins, to study their interactions or to control their activity in living cells. Incorporation of UAAs with bioorthogonal reactivity facilitates the site-specific installation of labels for spectroscopy and microscopy. Light-activatable crosslinker UAAs can be used to trap interacting molecules in living cells with a precision almost at the structural level. Post-translational modifications such as lysine acetylation and serine phosphorylation can be directly encoded to analyse their impact on protein function, and caging groups can be installed on critical residues to create light-activatable proteins. In this review we highlight recent applications of this technology to investigate protein function
Trapping Chromatin Interacting Proteins with Genetically Encoded, UV-Activatable Crosslinkers In Vivo
No full textOptimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes
No full textIncorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids
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