A 19-nucleotide RNA containing the CUGGGA loop sequence corresponding to nucleotides 30–35 of the HIV-1 trans-activation response element (TAR) was synthesized in vitro and analyzed by biochemical methods and one- and two-dimensional NMR spectroscopy. Diagnostic RNase cleavage patterns were similar for the loops in the full-length HIV-1 TAR and the 19-nucleotide RNA, indicating that they are similar in structure. NMR data showed that the loop is stabilized by base-stacking interactions. The first loop nucleotide is stacked upon the A-helical stem, and the loop uridine is stacked upon this cytosine. On the opposite side of the loop, the third loop guanosine is stacked upon the adenosine, which is stacked upon the stem. No specific Watson-Crick or non-Watson-Crick base pairing across the loop was identified. Unusually short interribose distances indicate a significant distortion of the sugar-phosphate backbone centered at the adenosine. Relatively short NMR relaxation times for protons of the adenosine and its adjacent guanosine, as well as rapidly exchanging imino protons, provide evidence for dynamic processes occurring in the loop.
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