Evolved Sequence Contexts for Highly Efficient Amber Suppression with Noncanonical Amino Acids

Abstract

The expansion of the genetic code with noncanonical amino acids (ncAA) enables the function of proteins to be tailored with high molecular precision. In this approach, the ncAA is charged to an orthogonal nonsense suppressor tRNA by an aminoacyl-tRNA-synthetase (aaRS) and incorporated into the target protein in vivo by suppression of nonsense codons in the mRNA during ribosomal translation. Compared to sense codon translation, this process occurs with reduced efficiency. However, it is still poorly understood, how the local sequence context of the nonsense codon affects suppression efficiency. Here, we report sequence contexts for highly efficient suppression of the widely used amber codon in E. coli for the orthogonal Methanocaldococcus jannaschii tRNA(Tyr)/TyrRS and Methanosarcina mazei tRNA(Pyl)/PylRS pairs. In vivo selections of sequence context libraries consisting of each two random codons directly up- and downstream of an amber codon afforded contexts with strong preferences for particular mRNA nucleotides and/or amino acids that markedly differed from preferences of contexts obtained in control selections with sense codons. The contexts provided high amber suppression efficiencies with little ncAA-dependence that were transferrable between proteins and resulted in protein expression levels of 70-110% compared to levels of control proteins without amber codon. These sequence contexts represent stable tags for robust and highly efficient incorporation of ncAA into proteins in standard E. coli strains and provide general design rules for the engineering of amber codons into target genes.

Re‐exploration of the Codon Context Effect on Amber Codon‐Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue–White Screening Assay

“…Three nucleotides upstream and downstream of the amber codon were randomised, and fused within the lacZ ‐α gene. Unlike a previous study that required multiple rounds of selection with quite a few read‐through clones obtained, thousands of clones were obtained here by one round of blue–white screening and easily distinguished by the depth of colour upon exposure to X‐gal . Large‐scale sequencing revealed a remarkable difference in downstream nucleotide preferences between blue and white clones.…”

“…Although the effect of codon context on read‐through has been extensively studied in yeast by screening a degenerate ADE2 library, little is known about this effect in Escherichia coli . Recently, a partial random library in which the fragment NNK‐NNK‐TAG‐NNK‐NNK was fused to the chloramphenicol acetyltransferase gene was used to explore codon context for NAA incorporation in E. coli . Based on the few clones that survived antibiotic selection, there was a nucleotide preference for low GC content and, in particular, for the presence of adenine at all N(nt) random positions and guanine or thymine balanced at the K(nt) random positions.…”

“…The codon-context effect has long been observed in nonsense translational read-throughb yho-mologouss uppressor tRNAs encoding canonical amino acids in prokaryotes [9] and eukaryotes; [10] this should also be considered for heterologous,o rthogonal tRNAs encoding NAAs.A lthought he effect of codon context on read-through has been extensively studied in yeastb ys creening ad egenerate ADE2 library, [11] little is known aboutt his effect in Escherichia coli.R ecently,apartial random libraryi nw hich the fragment NNK-NNK-TAG-NNK-NNK was fused to the chloramphenicol acetyltransferase gene was used to explore codon contextf or NAA incorporation in E. coli. [12] Based on the few clones that survived antibiotic selection, there was an ucleotide preference for low GC content and, in particular,f or the presence of adenine at all N(nt) random positions and guanineo rt hymine balanced at the K(nt) random positions. As trong overall preference for asparagine and lysine was observed at all positions.I t would be useful to explore ac omplete random library,a st he sequence surroundinga na mber codon usually interacts in a cooperative manner to influence translational read-through.…”

“…Three nucleotides upstream and downstream of the amberc odon were randomised, and fused within the lacZ-a gene. Unlike ap revious study [12] that required multiple rounds of selection with quite af ew read-through cloneso btained, thousands of clones were obtainedh ere by one round of blue-white screening and easily distinguishedb yt he deptho f colour upon exposure to X-gal. [14] Large-scale sequencing revealed ar emarkable difference in downstream nucleotide preferences betweenb lue and white clones.T here was moderate The effect of codon context on amber codon-guidedi ncorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection.H ere, we re-explored this effect by screening al ibrary in which three nucleotides upstream and downstream of the amber codon were randomised, andi nserted within the lacZ-a gene.…”

Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

“…A recent study examined this phenomenon by randomly varying the nucleotide sequences in the two codons immediately preceding (5′) and following (3′) the amber stop codon, and observing the affect on the incorporation efficiency of several UAAs expressed in E. coli (Pott, Schmidt, & Summerer, 2014). For pAcPhe incorporation, the authors found that the most efficient suppression occurred with the amino acid sequence Asn-Asn-TAG-Thr-Lys, with efficiencies for this optimized sequence approaching that of natural Tyr (no amber suppression) incorporation.…”