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 E. 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 the NCI/SAIC Protein Expression Laboratory, a highly efficient cell-free translation system has been coupled with a novel suppressor tRNA technology to site-specifically insert unnatural amino acid analogs into the p66 subunit of p66/p51 HIV-1 RT (Sitaraman et al., 2003). Using this approach (Klarmann et al., 2004), m-fluoro-Tyr and nor-Tyr were substituted for Tyr183 of the DNA polymerase -Tyr-Met-Asp-Asp- active site motif, the latter of which resulted in loss of RNA-dependent DNA polymerase while DNA-dependent DNA polymerase activity was unaffected. Subsequent to this, mercapto-Tyr, 1-naphthol-Tyr, 2-naphthol-Tyr, m-Tyr and methylamino-Tyr have been substituted for Tyr115 and Tyr183 of p66 RT. Preliminary data indicate that resistance to the nucleoside analog 3TC is induced following introduction of methylamino-Tyr at position 115. The ability to reconstitute and purify HIV-1 RT whose p66 subunit contains amino acid analogs will allow structure/function analysis to be performed at a level of resolution exceeding that obtained by conventional site-directed mutagenesis. Current efforts focus on the introduction of unnatural amino acids to study the role of stacking interactions between residues of HIV-1 RT and its nucleic acid substrate.[
| 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 |
