Substrate plasticity of the ribosomal biosynthetic machinery.

摘要

This thesis describes the reconstitution of a pure Escherichia coli translation system and the exploitation of the substrate plasticity of the protein biosynthetic machinery with synthetic acyl-tRNA substrates.; In Chapter 1, a review was given regarding the role of the amino acid played in each step of the protein biosynthesis and the tolerance of the translation machinery for different amino acid structures. A brief description of the substrate specificity of each step at the molecular level based on the recent high-resolution structures of the ribosomal machinery was provided, followed by the biochemical results with synthetic acyl-tRNA analogs for each step. We also look at the chemical literature for the unnatural amino acid incorporation in hope of identifying possibilities for expanding the substrate specificity of the ribosomal machinery through "ribosome engineering".; In Chapter 2 through Chapter 4, the development of genetic programming of peptidomimetic synthesis was described. Using a pure Escherichia coli translation system, we first showed that several adjacent, arbitrarily chosen sense codons can be completely reassigned to various unnatural amino acids according to de novo genetic codes by translating mRNAs into specific peptide analog polymers (peptidomimetics). By preventing competing reactions with aa-tRNA synthetases, aa-tRNAs, and release factors during translation and by using nonsuppressor aa-tRNA substrates, we tested the intrinsic specificity of the protein biosynthetic machinery for a series of amino acids with varied backbone structures. The results showed that different amino acids on the same tRNA adaptor give significantly different peptide yields, and the results also gave potential evidence for cross-talk between the amino acid and tRNA body/anticodon in aa-tRNA decoding by the ribosome. Using the same translation system, we also tested the possibility of ribosome catalyzed carbon-carbon bond formation reactions. These results further supported the substrate plasticity of the ribosomal biosynthetic machinery and provided immediate candidates for ribosomally encoded polymer synthesis.; In chapter 5, as a first step to "fix" poor analog substrates, we started to carry out mechanistic studies with our synthetic aa-tRNA substrates to determine whether the amino acid is simply being discriminated by EF-Tu binding or plays a more complex role in aa-tRNA decoding on the ribosome.