In collaboration with the NCI/SAIC Protein Expression Laboratory, a cell-free translation system was 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. In a subsequent study, we evaluated five HIV-1 RT variants containing tyrosine analogs at position 115 of their p66 subunit. Each mutant retained significant DNA polymerase activity, and two were selected for detailed kinetic analysis. Aminomethyl-Phe115 RT incorporated dCTP more efficiently compared to WT RT and is resistant to the chain-terminating nucleoside analog (-)-beta-2,3dideoxy-3-thiacytidine triphosphate (3TCTP). 2-Naphthyl-Tyr115 RT activity is significantly impaired at low dCTP concentrations and is kinetically slower with all dCTP analogs tested. Structural models of HIV-1 RT ternary complexes containing these amino acid substitutions that reveal probable mechanisms of the observed catalytic rate changes. Finally, genetically manipulated E.coli strains have been used to site-specifically introduce photoactivable amino acids for crosslinking to a variety of biomolecules.
| 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 |
