THE ROLE OF 2'-HYDROXYL GROUPS IN AN RNA-PROTEIN INTERACTION

The role of the 2'-hydroxyl group in RNA-protein interaction has been investigated using MS2 coat protein and its hairpin RNA operator as a model system. Derivatives of the MS2 translational operator were prepa red where individual riboses were replaced by deoxyribose and their bi nding affinities to MS2 coat protein were determined. Only 1 (U--5) ou t of 15 positions tested reduced protein affinity by 1.6 kcal/mol. A v ariety of other 2'-modifications were tested at this position to under stand the role of this particular 2'-hydroxyl group. Normal binding of the U-NH2 variant and weaker binding of the U-O-methyl variant are co nsistent with the ability of these functional groups to provide a hydr ogen bond donor. This is also supported by recent crystallographic dat a which indicate a possible interaction between the 2'-hydroxyl of U-- 5 and the carboxylate group of glutamate 63 [Valegard et al. (1994) Na ture 371, 623-626]. Complementary experiments introducing riboses into a DNA hairpin confirm the putative protein contact, and also identify a requirement for riboses in the two upper base pairs of the hairpin. Several arguments suggest these riboses are required to maintain an A -form helix in this region of the binding site. A minimum requirement of four 2'-hydroxyl groups for wild-type coat protein binding has been determined, one of which is at the-5 position and other three in the upper stem in any combination. The data are analyzed in terms of the r ecently determined structure of the free RNA by NMR [Borer et al. (199 5) Biochemistry 34, 6488-6503] and the cocrystal structure of the comp lex (Valegard et al., 1994.