Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

Ling, Jiqiang; O’Donoghue, Patrick; Söll, Dieter

doi:10.1038/nrmicro3568

Cited by 113 publications

(128 citation statements)

References 144 publications

(134 reference statements)

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“…But it is known in mitochondrial genomes, where a particular codon lost its original assignment and now leads to insertion of another amino acid [24] . Our case here is different; Sec insertion is mediated by a SECIS element and thus gives rise to dual use of the codon for another amino acid (through pairing with tRNA Sec variants carrying the proper anticodon).…”

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confidence: 99%

Facile Recoding of Selenocysteine in Nature

et al. 2016

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Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24,903 microbial genomes for tRNASec species. As expected, UGA is the predominant Sec codon in use. We also found tRNASec species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with 75Se or mass spectrometry. Other tRNASec species with different anticodons enabled Escherichia coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments. Our results reveal that the genetic code is much more flexible than previously thought.

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confidence: 99%

Facile Recoding of Selenocysteine in Nature

et al. 2016

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“…This code is preserved in virtually all life forms on earth with minor variations. 1 In recent years, the genetic code has been expanded to encode unnatural amino acids (Uaas) by introducing into live cells a new tRNA/aminoacyl-tRNA synthetase (aaRS) pair, 2,3 which is orthogonal to endogenous tRNA/aaRS pairs (Figure 1). The orthogonal aaRS is evolved to charge a desired Uaa onto the orthogonal tRNA, and the resultant Uaa-tRNA then incorporates the Uaa into proteins in response to a unique codon, such as the amber stop codon UAG.…”

Section: Introductionmentioning

confidence: 99%

Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

Wang

2017

Acc. Chem. Res.

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Conspectus Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E. coli, this methodology has now been expanded in multiple eukaryotic cells and animals. In this account, we focus on addressing various biological challenges for rewriting the genetic code, describing impacts of code expansion on cell physiology, and discussing implications for fundamental studies of code evolution. Specifically, a general method using the type-3 polymerase III promoter was developed to efficiently express prokaryotic tRNAs as orthogonal tRNAs and a transfer strategy was devised to generate Uaa-specific aaRS for use in eukaryotic cells and animals. The aaRSs have been found to be highly amenable for engineering substrate specificity toward Uaas that are structurally far deviating from the native amino acid, dramatically increasing the stereochemical diversity of Uaas accessible. Preparation of the Uaa in ester or dipeptide format markedly increases the bioavailability of Uaas to cells and animals. Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism of eukaryotic cells, degrades mRNA containing a premature stop codon. Inhibition of NMD increases Uaa incorporation efficiency in yeast and C. elegans. In bacteria, release factor one (RF1) competes with the orthogonal tRNA for the amber stop codon to terminate protein translation, leading to low Uaa incorporation efficiency. Contradictory to the paradigm that RF1 is essential, it is discovered that RF1 is actually nonessential in E. coli. Knockout of RF1 dramatically increases Uaa incorporation efficiency and enables Uaa incorporation at multiple sites, making it feasible to use Uaa for directed evolution. Using these strategies, the genetic code has been effectively expanded in yeast, mammalian cells, stem cells, worms, fruit flies, zebrafish, and mice. It is also intriguing to find out that the legitimate UAG codons terminating endogenous genes are not efficiently suppressed by the orthogonal tRNA/aaRS in E. coli. Moreover, E. coli responds to amber suppression pressure promptly using transposon insertion to inactivate the introduced orthogonal aaRS. Persistent amber suppression evading transposon inactivation leads to global proteomic changes with a notable up-regulation of a previously uncharacterized protein YdiI, for which an unexpected function of expelling plasmids is discovered. Genome integration of the orthogonal tRNA/aaRS in mice results in minor changes in RNA transcripts but no significant physiological impairment. Lastly, the RF1 knockout E. coli strains afford a previously unavailable model organism for st...

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“…The standard genetic code features 20 natural amino acids encoding 61 triplet sense codons, with the remaining three codons (TAA, TAG, and TGA) signaling translation termination [1]. In some organisms, certain TGA and TAG codons are recoded by selenocysteine (Sec) and pyrrolysine (Pyl), known as the 21 st and 22 nd natural amino acids, respectively [1–3]. Except for Sec, each of the 22 amino acids used as protein building blocks is ligated to the correct transfer RNA (tRNA) by a specialized aminoacyl-tRNA synthetase (aaRS) [4] (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Rewiring protein synthesis: From natural to synthetic amino acids

Fan

Evans

Ling

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. Scope of review This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. Major conclusions The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. General significance Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms.

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Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

Cited by 113 publications

References 144 publications

Facile Recoding of Selenocysteine in Nature

Facile Recoding of Selenocysteine in Nature

Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

Rewiring protein synthesis: From natural to synthetic amino acids

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