CONTACTS BETWEEN REVERSE-TRANSCRIPTASE AND THE PRIMER STRAND GOVERN THE TRANSITION FROM INITIATION TO ELONGATION OF HIV-1 REVERSE TRANSCRIPTION

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA s ynthesis. The initiation of reverse transcription requires specific in teractions between HIV-1 RNA, primer tRNA(3)(Lys) and RT, We have prev iously shown that extension of an oligodeoxyribonucleotide, a situatio n that mimicks elongation, is unspecific and differs from initiation b y the polymerization rate and dissociation rate of RT from the primer- template complex, Here, we used replication intermediates to analyze t he transition from the initiation to the elongation phases. We found t hat the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the poly merization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA(3)(Lys). The same increase in the polymeriz ation rate was observed when an oligoribonucleotide, but not an oligod eoxyribonucleotide, was used as a primer. In parallel, the dissociatio n rate of RT from the primer-template complex decreased 30-fold betwee n addition of the 17th and 19th nucleotide to tRNA(3)(Lys). The polyme rization and dissociation rates are most likely governed by interactio ns of the primer strand with helix alpha H in the p66 thumb subdomain and the RNase H domain of RT, respectively.