A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and Polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5′ end of the molecule, the stem contains a bulge at nucleotide C2, most likely a result of base pairing between G1 and C25. A method for distinguishing RNA stem-loops from palindromic dimers is described and was used to confirm that the EIAV TAR RNA has a stem-loop structure at conditions used for NMR spectroscopy. RNase A and RNase T1 cleavage patterns are consistent with the structural features derived from the NMR data.
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