ARGININE-BINDING RNAS RESEMBLING TAR IDENTIFIED BY IN-VITRO SELECTION

Specific binding of the human immunodeficiency virus Tar protein to it s RNA site (TAR) is mediated largely by a single arginine residue loca ted within a basic region of the protein. Many essential features of t he interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group b inds to a guanine base in the major groove and to two phosphates adjac ent to a bulge, with the RNA structure stabilized by a base triple bet ween a U in the bulge and an adjacent A:U base pair. To compare the TA R structure to other arginine-binding RNAs, we performed in vitro sele ction experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same mot if found in TAR: two stems separated by a bulge of at least two nucleo tides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G: C and A:U pairs, located next to small loops. Chemical modification ex periments demonstrated that these ''TAR-like'' RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outs ide the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-bindin g sites, we measured arginine-binding affinities of TAR-like RNAs havi ng all possible three-nucleotide loops. An RNA with a UAG loop bound w ith highest affinity, and chemical modification and RNase mapping expe riments suggested that the RNA changes conformation upon arginine bind ing, converting a large unstructured loop into a bulge conformation re lated to that of TAR. The results suggest that the arginine-binding si te in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.