Although in vitro site-directed mutagenesis has been invaluable in studying protein structure and function via targeted amino acid exchanges, this approach is limited inasmuch as only the other 19 amino acids defined by the genetic code can be introduced. In contrast, unnatural amino acids provide a rich source of agents to study protein structure and function by probing the space around an amino acid. Such expansion of the genetic code has been possible through engineering Escherichia coli to accept an orthogonal aminoacyl tRNA synthetase/tRNA pair, allowing the unusual amino acid to be introduced via translational suppression. Alternatively, coupled transcription/translation systems have been designed to exploit chemically charged tRNAs. In collaboration with SAIC-Frederick, we have established highly efficient methods for large-scale cell-free synthesis of p66/p51 HIV-1 reverse transcriptase (RT) (~1 mg) and, in collaboration with S. Hecht (University of Virginia), have successfully introduced 7-azatryptophan and 2-napthylalanine into p66 of the p66/p51 heterodimer. Future efforts will use this strategy to provide a detailed biochemical analysis of residues critical to the DNA polymerase and RNase H functions of HIV-1 RT. Examples include (a) residues conferring resistance to nucleoside analogs (Tyr215), (b) conserved carboxylate residues comprising the RNase H catalytic center (D443, E478, D498, and D549) and (c) residues contributing to specialized RNase H functions (e.g., Tyr501, which controls PPT selection, sensitivity to RNase H inhibitors, and the fidelity of DNA synthesis). A long-term goal of this project is 19F-NMR analysis of fluorotyrosine-substituted enzymes.
| Rausch, Jason W; Le Grice, Stuart F J (2007) Exploiting structurally diverse nucleoside analogs as probes of reverse transcription complexes. Curr HIV Res 5:11-22 |
| Rausch, Jason W; Le Grice, Stuart F J (2007) Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis. Nucleic Acids Res 35:256-68 |
| Sitaraman, Kalavathy; Esposito, Dominic; Klarmann, George et al. (2004) A novel cell-free protein synthesis system. J Biotechnol 110:257-63 |
| Klarmann, George J; Eisenhauer, Brian M; Zhang, Yi et al. (2004) Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase. Protein Expr Purif 38:37-44 |
