An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli.

BACKGROUND: Adding new amino acids to the set of building blocks for protein synthesis expands the scope of protein engineering, and orthogonal pairs of tRNA and aminoacyl-tRNA synthetase have been developed for incorporating unnatural amino acids (UAAs) into proteins. While diverse systems have been developed to incorporate UAAs in response to the amber codon, less research has been focused on four-base codons despites their advantages. In this study, we report an efficient method to incorporate UAA in response to an AGGA codon in Escherichia coli. RESULTS: The Methanococcus jannaschii tyrosyl-tRNA synthetase-tRNACUA(MjTyrRS-MjtRNACUA) orthogonal pair has been engineered to incorporate diverse UAAs in response to the amber codon. To apply the engineered MjTyrRS enzymes for UAAs to a four-base codon suppression, we developed an MjTyrRS-MjtRNAUCCU pair system that enabled incorporation of UAAs in response to the AGGA codon in E. coli. Using this system, we demonstrated that several UAAs could be incorporated quantitatively in the AGGA site. In addition, multiple AGGA codons were successfully suppressed in an E. coli strain when the endogenous tRNACCUArg gene was knocked out. CONCLUSION: An efficient system was developed for the incorporation of UAAs in response to the AGGA four-base codon in E. coli, and the method was successfully demonstrated for several UAAs and for multiple AGGA sites. GENERAL SIGNIFICANCE: The developed system can expand the repertoire of protein engineering tools based on amino acid analogues in combination with other UAA incorporation methods. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Rewiring protein synthesis: From natural to synthetic amino acids.

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. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Absence of effect of varying Thr-Leu codon pairs on protein synthesis in a T7 system.

The over-represented threonine-leucine (Thr-Leu) codon pair ACG CUG has been previously reported to be inhibitory to translation compared to the synonymous under-represented Thr-Leu codon pair ACC CUG, in an E. coli system in which the codon pairs were located either 3 and 4, or 6 and 7, or 9 and 10 codons downstream from the initiating codon for the message [Irwin, B., Heck, J. D., and Hatfield, G. W. (1995) J. Biol. Chem. 270, 22801-22806]. In the work reported here, these synonymous codon pairs were tested in a T7 system, with the codon pairs located either 14 and 15, or 6 and 7 codons downstream from the AUG start codon. In contrast to the reported findings in the E. coli system, there was no difference found in translation between mRNAs containing the respective codon pairs in the T7 system. The reasons for the different findings remain unclear, but presumably are a consequence of differences between the E. coli and T7 systems used to assay gene expression. Nevertheless, as a result of this work, it appears that the effect of varying codon pairs reported in the E. coli system is not due to a difference in translational step times through the respective codon pairs, as previously proposed.

[Synthesis of leucine codons and their use in the study of tRNALeu codon correspondence]. / Sintez kodonov leitsina i ikh ispol'zovanie dlia izucheniia kodovogo sootvetstviia tRNKLeu.

The leucine codon--UUA, UUG, CUU, CUC, CUA, CUG--was prepared via the phosphotriester approach. The purity of codon was confirmed by reverse phase chromatography in micro scale. The absence of migration of internucleotidic linkages was confirmed by hydrolysis with pancreatic RNase. Synthetic codon were used to study the codon response of six isoacceptor tRNALeu from the cow mammary gland.