Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine

Steinfeld, Justin B.; Aerni, Hans R.; Rogulina, Svetlana; Liu, Yuchen; Rinehart, Jesse

doi:10.1021/cb5000532

Cited by 35 publications

(49 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S5 and S6) are in moderate agreement with the enzyme-kinetic data. However, aminoacylation is only one component that influences translation efficiency; ncAA uptake (44) and stability and elongation factor Tu (EF-Tu) compatibility (7) are known to alter the efficiency of translation. It is also possible that some ncAAs or translation of offtarget UAG codons could alter the cellular proteome or metabolism in ways that may have an impact on protein synthesis and its fidelity.…”

Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

146

View full text Add to dashboard Cite

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

show abstract

Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

146

View full text Add to dashboard Cite

show abstract

“…Efficiency of site-specific incorporation of ncAAs via nonsense suppression depends on the catalytic prowess of the OTS [ 3 ], the availability of ncAA (depending on the ncAA intake and its participation in the cellular metabolism, [ 45 ]), the suitability of ncAA-o-tRNA for EF-Tu binding [ 12 ], and the capacity of ncAA-o-tRNA to decode the stop codon of interest [ 26 ] and outcompete the release factor for binding to the same site [ 46 ]. Because each of these processes is usually less efficient than for endogenous translation systems, yields of ncAA-containing proteins are typically low.…”

Section: Resultsmentioning

confidence: 99%

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

et al. 2018

View full text Add to dashboard Cite

Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

show abstract

“…For example, incorporation of phosphoserine into human MEK1 (mitogen-activated ERK activating kinase 1) in E. coli was achieved by deletion of serB , encoding phosphoserine phosphatase SerB [ 25 ]. To further increase the intracellular concentration of phosphoserine, Δ serB cells were grown in low or high phosphate minimal media [ 26 ]. In the low phosphate media, the PHO (phosphate) regulon is induced [ 27 ], which stimulates the uptake of phosphoserine by PhnE [ 26 ].…”

Section: Engineering Of Translation Components For the Improvementmentioning

confidence: 99%

“…To further increase the intracellular concentration of phosphoserine, Δ serB cells were grown in low or high phosphate minimal media [ 26 ]. In the low phosphate media, the PHO (phosphate) regulon is induced [ 27 ], which stimulates the uptake of phosphoserine by PhnE [ 26 ]. On the other hand, in the high phosphate medium, the degradation of phosphoserine by PHO regulon is suppressed [ 26 ].…”

Section: Engineering Of Translation Components For the Improvementmentioning

confidence: 99%

“…In the low phosphate media, the PHO (phosphate) regulon is induced [ 27 ], which stimulates the uptake of phosphoserine by PhnE [ 26 ]. On the other hand, in the high phosphate medium, the degradation of phosphoserine by PHO regulon is suppressed [ 26 ]. Under these optimized conditions, intracellular level of phosphoserine was elevated at a comparable concentration to those of canonical amino acids, and incorporation of phosphoserine into protein was improved.…”

Section: Engineering Of Translation Components For the Improvementmentioning

confidence: 99%

See 1 more Smart Citation

Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

Terasaka

Iwane

Geiermann

et al. 2015

IJMS

View full text Add to dashboard Cite

Genetic code expansion and reprogramming methodologies allow us to incorporate non-canonical amino acids (ncAAs) bearing various functional groups, such as fluorescent groups, bioorthogonal functional groups, and post-translational modifications, into a desired position or multiple positions in polypeptides both in vitro and in vivo. In order to efficiently incorporate a wide range of ncAAs, several methodologies have been developed, such as orthogonal aminoacyl-tRNA-synthetase (AARS)–tRNA pairs, aminoacylation ribozymes, frame-shift suppression of quadruplet codons, and engineered ribosomes. More recently, it has been reported that an engineered translation system specifically utilizes an artificially built genetic code and functions orthogonally to naturally occurring counterpart. In this review we summarize recent advances in the field of ribosomal polypeptide synthesis containing ncAAs.

show abstract

Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine

Cited by 35 publications

References 31 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

Contact Info

Product

Resources

About