Virginia Cornish
Helena Rubinstein Professor
Department of Chemistry

work : +1 212-854-5209

Cornish Group

Research: Ribosome Chemistry

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The ribosome is a large complex of RNA and protein that catalyzes the translation of RNA into protein.  The ribosome is impressive in its ability to convert between nucleic acid and peptide, two structurally unrelated polymers.  In addition, unlike most natural enzymes, the ribosome shows broad substrate specificity and uses amino acids with diverse chemical side chains.  The adaptor hypothesis suggests that the amino acid specificity of the ribosome should be entirely determined by the tRNA anticodon and independent of amino acid structure.  Several laboratories have now shown in fact that suppressor tRNAs and unnatural base pairs can be used to add a "21st amino acid" to the genetic code.  Rather than adding on to the genetic code, we set out to show that the genetic code could instead be "rewritten" using synthetic aminoacyl-tRNAs and a purified in vitro translation system.  This project is in collaboration with Prof. Steve Blacklow and Dr. Tony Forster at Harvard Medical School.  We have now completed a proof-of-principle for this strategy, demonstrating that we can synthesize a peptide containing three different unnatural amino acids in a row, each coded by a different, natural RNA codon.  This research was published this year in Proc. Natl. Acad. Sci. USA and featured in Chem. Biol.  The long-term goal of this project is to study the mechanism of translation and to manipulate the ribosome to catalyze polymers other than polypeptides.  Currently, we are focused on adapting the purified translation system for ribosome display of peptidomimetics and testing the adaptor hypothesis.

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Miniature Genetic Code

[Published in A. Forster, Z. Tan, M. N. L. Nalam, H. Lin, H. Qu, V.W. Cornish, S. Blacklow.  "Programming peptidomimetic syntheses by translating genetic codes designed de novo."  Proc. Natl. Acad. Sci. USA, 100, 6353-6357 (2003).  Featured in Chem. Biol., 10, 586-587 (2003) and in Chem. & Eng. News, 82, 64-68 (2004).]

A purified translation system should allow all 64 codons to be recognized by synthetic aminoacyl-tRNAs because there are no contaminating aminoacyl-tRNA synthetases that would otherwise proofread and recharge the tRNAs with the natural amino acids.  To test this hypothesis, the purified translation system was used to synthesize a short peptide with three unnatural amino acids in a row--each coded by a different natural codon (Asn, Thr, and Val).  Translation reactions showing that product formation was dependent on all three synthetic aminoacyl-tRNAs established that there was no cross-reactivity.  Co-migration of radiolabeled translation product with authentic peptide prepared by solid-phase synthesis on C18 reverse-phase HPLC further confirmed these results.  This experiment demonstrates the feasibility of using different synthetic aminoacyl-tRNAs as substrates for all 64 codons in the genetic code.

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Ribosome Display

[A.C. Forster, V.W. Cornish, S.C. Blacklow.  "Pure Translation Display." Anal. Biochem., 333, 358-364 (2004).]

Ribosome display allows very large libraries (1012-1015) of peptides to be synthesized and selected for function because the peptides can be linked to their unique RNA sequences in vitro.  Here we use the pure translation system for ribosome display, selecting for a biotinyl-amino acid via avidin beads. Back to Top  

Mechanistic Investigations

[Z. Tan, A. Forster, S. Blacklow, V.W. Cornish.  "Amino acid backbone specificity of the Escherichia coli translation machinery," J. Am. Chem. Soc., 126, 12752-12753 (2004).]

The adaptor hypothesis predicts that aa-tRNA decoding on the ribosome depends solely on the tRNA anticodon and is independent of the amino acid structure.  Here we look at the ability of the E. coli ribosomal biosynthetic machinery to accept aa-tRNA substrates with different amino acid analogs, but the same tRNA body and anticodon as a test of this hypothesis.

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