Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism

Vincent, Russel M.; Wright, Bradley W.; Jaschke, Paul R.

doi:10.1021/acssynbio.9b00021

Cited by 16 publications

(13 citation statements)

References 42 publications

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“…Thea bove data demonstrate initiation with ncAAs using itRNA Ty 2 albeit with relatively low efficiency.s fGFP-2am production is similar with itRNA Ty2 or Mj-tRNA Ty r (Figure S3), suggesting that inefficient initiation is not due to poor activity of Mj-TyrRS towards itRNA Ty 2 .Recently,the cellular effects of initiation with Met at UAGcodons were thoroughly characterized in ag enomically recoded organism (GRO) engineered for improved amber suppression. [9] We measured sfGFP expression in two GROs but observed improvements only in elongation ( Figure S13). This suggests that inefficient initiation is not due to competition with release factor 1, which is deleted in these strains.…”

Section: Communicationsmentioning

confidence: 99%

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

Tharp

Amikura

et al. 2020

Angew Chem Int Ed

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By transplanting identity elements into E. coli tRNAfMet, we have engineered an orthogonal initiator tRNA (itRNATy2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

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Section: Communicationsmentioning

confidence: 99%

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

Tharp

Amikura

et al. 2020

Angew Chem Int Ed

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“…On average across the strains we saw an 88-fold increase in fluorescence upon tRNA 2 expression with MG1655 showing the smallest increase and C122 showing the largest increase. As noted previously [1], the combination of lack of expression when repressed, along with a large increase after tRNA induction, could be useful for conditionally expressing toxic proteins due to lack of translational leakage from the UAG stop codon in the absence of the tRNA 2 .…”

Section: Resultsmentioning

confidence: 84%

“…We speculate that tRNA 2 expression has caused an increase in the cellular concentration of ribosomes and this has resulted in increased translation from the sfGFP(AUG) reporter. Support for this idea comes from a recent paper showing evidence that expressing initiator tRNA in E. coli increases mature ribosome levels and accelerates rRNA processing [28] and our recent work showing the amber initiator can increase ribosome protein abundance in cells [2]. We can speculate that expressing tRNA 2 has a similar effect on ribosomal maturation and rRNA processing in the cell, considering the presence of identical identity elements (aside from the anticodon sequence) in both initiator tRNAs.…”

Section: Resultsmentioning

confidence: 87%

“…Differences in basal levels of amber suppression within common K-12 strains is already known [7]. Expressing the amber initiator tRNA shows great promise as a way of programming orthogonal translation initiation at UAG sites in engineered genetic constructs [2], but Matters (ISSN: 2297-8240) | 5 [14]. See figure S1 for Mauve whole genome alignment between E. coli strains in green [15] [16].…”

Section: Introductionmentioning

confidence: 99%

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An orthogonal amber initiator tRNA functions similarly across diverse <em>Escherichia coli</em> laboratory strains

Vincent

Yiasemides

Jaschke

2019

Matters

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Translation initiation is a sequential process involving interactions between the 30S small ribosomal subunit, initiation factors and initiator tRNA. The Escherichia coli K-12 strain is unique in the Escherichia because it has two different initiator tRNA sequences, tRNA fMet1 encoded by the metZWV genes and tRNA fMet2 encoded by the metY gene. A mutant of the metY gene was previously made where the anticodon sequence, responsible for specifying the start codon where translation initiation begins, was changed so that it bound to the amber stop codon UAG instead of the usual AUG start codon [1]. This amber initiator tRNA has already been shown to be functional in the K-12 strain [1] [2], but it is unclear whether it would function in other strains normally lacking the tRNA fMet2 variant. In this work, we transformed E. coli K-12, and four other generally regarded as safe (GRAS) laboratory strains, with a plasmid expressing the amber initiator tRNA and evaluated its functionality and growth effects on the bacteria. We performed these tests because, despite these strains all belonging to E. coli phylogenetic group A, it is well known that there is significant variation between even closely related E. coli strains in their metabolism [3] [4] [5], transcriptional response to exogenous DNA expression [6] and rates of amber stop codon suppression [7]. We found that the amber initiator functions similarly across the five strains, effectively initiating translation at the orthogonal UAG start codon and that it had modest growth-slowing effects in the Crooks, W, and K-12 strains. The five tested E. coli strains in this work (K-12, B, C, W, and Crooks) are important workhorses of academic and industrial research and development. The path is now clear to deploy the amber initiator tRNA into these five strains to precisely control gene expression.

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“…The efficiency and orthogonality of amber codon initiation has since been demonstrated in several common laboratory strains of E. coli and in Mycobacterium smegmatis . Recently the effects of amber‐reader tRNA fMet (tRNA fMet CUA ) expression were thoroughly characterized in a genomically recoded organism in which all occurrences of UAG were removed from the genome . Although tRNA fMet CUA expression was found to upregulate expression of proteins associated with tRNA degradation and amino acid biosynthesis, as well as ribosome‐associated proteins, proteomic analyses provided no evidence for off‐target translation initiation.…”

Section: Non‐aug Initiationmentioning

confidence: 99%