Background: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extremely helpful. Often the question arises whether conformations obtained in this way appropriately reflect the reactivity of enzymes, however. Rates that characterize transitions are therefore compulsory experiments for the elucidation of the reaction mechanism. Such experiments have been performed for the reverse transcriptase of the type 1 human immunodeficiency virus (HIV-1 RT).
Results: We have developed a methodology to monitor the interplay between HIV-1 RT and its DNA substrate. To probe the protein-DNA interactions, the sugar backbone of one nucleotide was modified by a substituent that influenced the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide follows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar substituent and the enzyme. The model was confirmed by kinetic experiments with HIV-1 RT mutants.
Conclusions: Experiments with HIV-1 RT demonstrate that strand-elongation efficiency using a modified nucleotide correlates well with distances between the DNA substrate and the enzyme. The functional group at the modified nucleotides acts as an 'antenna' for steric interactions that changes the optimal transition state. Kinetic experiments in combination with backbone-modified nucleotides can therefore be used to gain structural information about reverse transcriptases and DNA polymerases.