Rewiring translation – Genetic code expansion and its applications

Abstract

a b s t r a c tWith 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/tRNA CUA 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.

A Cascade of Histone Modifications Induces Chromatin Condensation in Mitosis

“…Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) pathway, which functions as an adaptive immune system in bacteria (13), has been co-opted to engineer mammalian genomes in an efficient manner (14)(15)(16). In this two-component system, a single-guide RNA (sgRNA) directs the Cas9 nuclease to cause double-stranded cleavage of matching target DNA sequences (17).…”

“…However, all evidence that this interaction mediates chromosome condensation during mitosis is indirect. Here, we inserted the ultraviolet light (UV)-inducible cross-linker amino acid p-benzoyl-Lphenylalanine (pBPA) in histone proteins of living yeast, using genetic code expansion (13,14) to trace the dynamic interactions of these proteins along the cell cycle. We expressed Escherichia coli Tyr-tRNA CUA and its cognate pBPA-specific tRNA synthetase in Saccharomyces cerevisiae cells (15), leading to the incorporation of pBPA in response to amber codons in an additional, plasmid-borne histone H2A gene [tagged with hemagglutinin (HA) and under its native promoter].…”

Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

“…Strategies to genetically engineer strains for efficient conversion of carbon sources into the desirable end product in a growth-uncoupled fashion have been demonstrated for L. lactis for the production of its endogenous metabolites (84)(85)(86)(87) and the alternative metabolite L-alanine (18). Moreover, retentostat cultures provide an excellent test bed for quantitative analysis of radical synthetic biology strategies aimed at uncoupling native and heterologous protein production by introduction of orthogonal translation systems (88,89). Also, retentostats are not the only means to uncouple growth and anabolic product formation.…”

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

“…[11][12][13][14][15][16][17][18][19][20] The caged amino acids are inserted in response to an amber stop codon, TAG, by suppressor tRNAs that have been misacylated by engineered tRNA synthetases. [21][22][23][24][25][26][27][28] Advantages of this methodology over other approaches are that the location of the unnatural amino acid is determined through genetic mutagenesis and the light-activated proteins are produced in live cells eliminating the requirement for transfection or injection. The addition of non-mammalian RNA polymerases to the genetic circuitry of cells enables the development of orthogonal genetic expression platforms that can be manipulated to activate specific genes that will not be expressed by the endogenous cellular genetic machinery.…”