Unnatural Amino Acids as Probes of RT Structure and Func

Basic Sciences

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Abstract

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.