The ribose 2′-hydroxyl group confers upon RNA many unique molecular properties. To better appreciate its contribution to structure and stability and to monitor how substitutions of the 2′ hydroxyl can alter an RNA molecule, each loop pyrimidine ribonucleotide in the UUCG tetraloop was substituted with a nucleotide containing either a fluorine (2′-F), hydrogen (2′-H), amino (2′-NH2), or methoxy (2′-OCH3) group, in the context of both the C:G and G:C loop-closing base pair. The thermodynamic parameters of these tetraloop variants have been determined and NMR experiments used to monitor the structural changes resulting from the substitutions. The modified riboses are better tolerated in the G[UUCG]C tetraloop, which may be due to its increased loop flexibility relative to the C[UUCG]G loop. Even for these simple substitutions, the free-energy change reflects a complex interplay of hydrogen bonding, solvation effects, and intrinsic pucker preferences of the nucleotides.

Tryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N-terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1–101; the C-terminal RBD2, residues 202–282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the β-sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady-state and time-resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the β-sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3′ UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar–phosphate backbone in the RNA–RBD complexes. RBD1(K20C)–EPD-Fe cleaves the 5′ strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5′ side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.