Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

Wang, Kaihang; Neumann, Heinz; Peak-Chew, Sew Y.; Chin, Jason W.
July 2007
Nature Biotechnology;Jul2007, Vol. 25 Issue 7, p770
Academic Journal
In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1–mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNACUA-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from ∼ 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.


Related Articles

  • An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Terasaka, Naohiro; Hayashi, Gosuke; Katoh, Takayuki; Suga, Hiroaki // Nature Chemical Biology;Jul2014, Vol. 10 Issue 7, p555 

    The Watson-Crick base pairs between the 3′-terminal end of tRNAs and ribosomal RNA in the peptidyl transferase center are universally conserved. Here, we report that the introduction of compensatory mutations to Escherichia coli RNAs in this site leads to an orthogonal system independent...

  • Molecular biology: Inseparable ribosomes. Rusk, Nicole // Nature Methods;Oct2015, Vol. 12 Issue 10, p913 

    The article presents a study on the function and engineering possibilities of fusing ribosomal subunits. The study shows that the small subunit binds first to the messenger (m)RNA's Shine-Dalgarno sequence during translation. It reveals that the larger subunit catalyzes and associates the...

  • Chemical biology: Building blocks for peptide drugs. Owens, Joanna // Nature Reviews Drug Discovery;Jun2004, Vol. 3 Issue 6, p476 

    The discovery of novel peptide drugs could he simplified by a new protocol for building diverse, messenger RNA (mRNA)-encoded peptide libraries. This strategy has been hindered previously by the difficulty of generating sufficient amounts of tRNAs bearing diverse, non-standard amino acid...

  • Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host. Angov, Evelina; Hillier, Collette J.; Kincaid, Randall L.; Lyon, Jeffrey A. // PLoS ONE;2008, Vol. 3 Issue 5, p1 

    Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between...

  • Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Neumann, Heinz; Wang, Kaihang; Davis, Lloyd; Garcia-Alai, Maria; Chin, Jason W. // Nature;3/18/2010, Vol. 464 Issue 7287, p441 

    The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA) and the Methanosarcina barkeri pyrrolysyl-tRNA...

  • Broad-Specificity mRNA-rRNA Complementarity in Efficient Protein Translation. Barendt, Pamela A.; Shah, Najaf A.; Barendt, Gregory A.; Sarkar, Casim A. // PLoS Genetics;Mar2012, Vol. 8 Issue 3, Special section p1 

    Studies of synthetic, well-defined biomolecular systems can elucidate inherent capabilities that may be difficult to uncover in a native biological context. Here, we used a minimal, reconstituted translation system from Escherichia coli to identify efficient ribosome binding sites (RBSs) in an...

  • Two isoforms of Rubisco activase in cotton, the products of separate genes not alternative splicing. Salvucci, Michael E.; van de Loo, Frank J.; Stecher, Dawn // Planta;Mar2003, Vol. 216 Issue 5, p736 

    In several plant species, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA. Two forms of activase corresponding to the longer, redox-regulated α and the shorter, β forms were detected...

  • Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor. Beck, Konstanze; Wu, Long-Fei; Brunner, Josef; Müller, Matthias // EMBO Journal;1/1/2000, Vol. 19 Issue 1, p134 

    Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking ht vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein...

  • Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli. Liu, Dan-Feng; Ding, Hai-Tao; Du, Yi-Qing; Zhao, Yu-Hua; Jia, Xiao-Ming // Applied Biochemistry & Biotechnology;Apr2012, Vol. 166 Issue 5, p1301 

    A phosphite dehydrogenase gene ( ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics