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Orthogonal Ribosome–tRNAs Pair by Engineering of Peptidyl Transferase Center

  • Naohiro TerasakaEmail author
Chapter
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Part of the Springer Theses book series (Springer Theses)

Abstract

The CCA-3′ sequence of tRNA is conserved among all organisms and is important for aminoacylation and translation. In bacteria, the CCA-3′ end makes Watson–Crick base pairs with bases of 23S rRNA in the peptidyl transferase center (PTC) in the classical state (Voorhees et al. in Nat Struct Mol Biol 16:528–533, 2009) and translocation (Dorner et al. in Nat Struct Mol Biol 13:234–241, 2006) during translation. These base pairs are important for translation activity, and the compensatory mutations in these base pairs are tolerated during peptidyl transfer reaction (Kim and Green in Molecular Cell 4(5):859–864, 1999) and translocation (Dorner et al. in Nat Struct Mol Biol 13:234–241, 2006). However, it is yet empirically unknown whether such mutations accommodates translation in its entirety. I will report development of orthogonal translation machinery (Terasaka et al. in Nat Chem Biol 10(7):555–557, 2014). First, I developed a method to easily aminoacylate various amino acids onto tRNAs bearing mutation (or mutations) in CCA-3′ end using compensatory mutated flexizymes. Then the translation activity of the PTC-mutated ribosomes and tRNAs were measured. Consequently, I developed the mutant ribosome–tRNA pair which had comparable translation activity and orthogonality to the wild-type ribosome–tRNA pair. Finally, two different peptides were expressed simultaneously in one pot from a single mRNA template using the mutant and wild-type ribosome–tRNA pairs.

Keywords

tRNA Ribosome Translation Peptide Synthetic biology 

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Copyright information

© Springer Japan KK 2017

Authors and Affiliations

  1. 1.ETH ZurichZurichSwitzerland

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