MAJOR GROOVE OPENING AT THE HIV-1 TAT-BINDING SITE OF TAR RNA EVIDENCED BY A RHODIUM PROBE

Transactivation of human immunodeficiency virus (HIV) gene expression requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. The TAR RNA contains a six-nucleotide loop and a three-nucleotide pyrimidine bulge which separates two helical stem regions. The trinucleotide bulge is essential for high affinity and specific binding of the Tat protein. Recently, a rhodium complex, Rh(phen)(2)phi(3+), was discovered which promotes RNA cleavage in the open major groove and triply bonded bases [Chow, C. S., ed al. (1992) Biochemistry 31, 972-982]. This metal complex does not bind double-helical RNA or unstructured single-stranded regions of RNA. Instead, sites of tertiary interaction which are open in the major groove and accessible to stacking are targeted by the complex through photoactivated cleavage. We have used this rhodium probe to investigate the effect of bulge bases on the major groove opening in TAR RNA. The sites targeted by the rhodium complex have been mapped to single nucleotide resolution on wild-type TAR RNA and on several mutants of the TAR RNA containing different numbers of mismatch bases in the bulge region. A strong cleavage at residues C39 and U40 was observed on the wild-type TAR RNA and in mutant TAR RNA containing two mismatch bases in the bulge. No cleavage at C39 and U40 was observed in a bulgeless and a one-base bulge TAR RNA. By varying the number of mismatch bases, we demonstrated that the trinuclear bulge widens the major groove of TAR RNA to facilitate Tat binding. Our studies establish three important factors involved in Tar-TAR recognition: (i) A bulge containing two or more bases permits major groove widening sufficient for Tat binding, and this cannot be accomplished by a single-base bulge; (ii) Tat fragment (42-72) binds in the major groove of TAR RNA because cleavage at C39 and U40 in wild-type TAR RNA was inhibited in the presence of Tat(42-72); (iii) cleavage efficiency is unaffected by the presence of arginine, indicating that the conformational changes in TAR RNA upon arginine binding have no effect on the widening and accessibility of the major groove.