MAPPING OF A PROTEIN-RNA KISSING HAIRPIN INTERFACE - ROM AND TAR-TAR

An RNA 'kissing' complex is formed by the association of two hairpins via base pairing of their complementary loops, This sense-antisense RN A motif is used in the regulation of many cellular processes, includin g Escherichia coli ColE1 plasmid copy number, The RNA one modulator pr otein (Rom) acts as a co-regulator of ColE1 plasmid copy number by bin ding to the kissing hairpins and stabilizing their interaction. We hav e used heteronuclear two-dimensional NMR spectroscopy to map the inter face between Rom and a kissing complex formed by the loop of the trans -activation response (Tar) element of immunodeficiency virus 1 (HIV-1) and its complement. The protein binding interface was obtained from c hanges in amide proton signals of uniformly N-15-labeled Rom with incr easing concentrations of unlabeled Tar-Tar. Similarly, the RNA-bindin g interface was obtained from changes in imino proton signals of unifo rmly N-15-labeled Tar with increasing concentrations of unlabeled Rom, Our results are in agreement with previous mutagenesis studies and pr ovide additional information on Rom residues involved in RNA binding. The kissing hairpin interface with Rom leads to a model in which the p rotein contacts the minor groove of the loop-loop helix and, to a less er extent, the major groove of the stems.