Bioconjugation can be defined as site-specific tethering of two molecules to generate a novel complex displaying the combined properties of its individual components. Although in its infancy, this discipline is rapidly emerging as a powerful complement to high-resolution crystallographic and spectroscopic methods in providing structural information on protein-nucleic acid complexes. Since the first study attaching the chemical nuclease 5-(iodoacetyl)-1,10-phenanthroline to Cys178 of the Escherichia coli catabolite gene activator protein (CAP), photoactivable, fluorescent, nucleolytic, and proteolytic agents have been site-specifically tethered to both the protein and nucleic acid components of nucleoprotein complexes. The Le Grice laboratory is currently employing biocongugate strategies to understand the interaction of p66/p51 HIV reverse transcriptase (RT) with its conformationally distinct nucleic acid substrates (duplexes of DNA and RNA, as well as RNA-DNA hybrids) and accessory viral proteins that have been shown to stimulate reverse transcription. The illustration summarizes regions of contact between the C-terminal ribonuclease H (RNase H) domain of p66 HIV-1 RT and bases of template and primer, revealed by crosslinking of an enzyme to which p-azidophenacyl was site-specifically tethered to the RNase H domain.

Last modified: 19 December 2008