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.