Physical mapping of HIV reverse transcriptase to the 5 ' end of RNA primers

Enzymatic analysis of RNA cleavage products has suggested that human immuno deficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RN As that are recessed on a longer DNA template (RNA primers) yet binds to th e 3' end of DNA primers. One concern is that RT molecules bound at the 3' e nd of RNA would not be easily detected because RT may not catalyze substant ial RNA extension or cleavage when bound to the 3' end. We used physical ma pping to show that RT binds preferentially to the 5' end of RNA primers. Am HIV-RT that lacked RNase H activity (HIV-RTE478Q) was incubated with the R NA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT prot ected a similar to 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal s tructure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wildtype HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.