Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Tian, Hong; Deng, Danni; Jie, Huang; Yao, Dongning; Xu, Xiaowei; Gao, Xiangdong

doi:10.1016/j.biochi.2012.12.010

Cited by 15 publications

(17 citation statements)

References 22 publications

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“…On the other hand, species-specific toxicity of the amber suppressor tRNA was also found (51). The usefulness of translational switches could depend on the host species.…”

Section: Discussionmentioning

confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-L-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-L-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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“…On the other hand, species-specific toxicity of the amber suppressor tRNA was also found (51). The usefulness of translational switches could depend on the host species.…”

Section: Discussionmentioning

confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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“…Synthetic life forms are steadily expanding: genome-wide replacements of Trp by 4FTrp and 6FTrp have been achieved with E. coli B7-3 and bacteriophage [ 186 , 187 , 188 ], and replacement of Trp by l -β-(thieno[3,2- b ]pyrrolyl)alanine ([3,2]Tpa) in E. coli MT20 [ 189 ]. Moreoever, based on the finding that numerous eARS are domain-specific and display strikingly low reactivities toward tRNA from the other side of a schism between the Bacteria and Archaea-Eukarya blocs [ 190 ], the use of orthogonal eARS-tRNA pairs, e.g., archaeal TyrRS-tRNATyr from Mja, LeuRS-tRNALeu from Mth or GluRS from Pho with a consensal archaeal tRNAGlu has enabled the incorporation of NCAA into specific sites of the proteome in both prokaryotes and eukaryotes [ 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 , 200 , 201 , 202 , 203 , 204 , 205 , 206 , 207 , 208 , 209 , 210 ], as has been attained through phenotypic suppression [ 211 ]. The DNA alphabet has been altered as well, where the canonical deoxyribonucleotide (CDN) thymine is replaced by the noncanonical deoxyribonucleotide (NCDN) 5-chlorouracil, yielding both optional and mandatory synthetic life forms [ 212 , 213 , 214 ] ( Table 4 ).…”

Section: Origin Of Synthetic Lifementioning

confidence: 99%

Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life

Wong

Mat

et al. 2016

Life

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The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets.

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“…These observations agree well with the result that anticodons corresponding to stop codons were less toxic or non-toxic. However, species-specific toxicity of stop codon suppression still needs to be considered carefully [ 63 ].…”

Section: Discussionmentioning

confidence: 99%

An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance

Kato

2021

Life

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We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by “informational disturbance”, in which the highly conserved system for transmission of information from DNA to proteins is perturbed.

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Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Cited by 15 publications

References 22 publications

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life

An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance

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