Crystal structure of an RNA duplex r(gug [formula omitted]cgcac) 2 with uridine bulges

Abstract

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gug u cgcac) 2, was determined at 1.4 Å resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R work/ R free of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4 122 with two independent molecules in the asymmetric unit. The unit cell dimensions are a = b = 47.18 Å and c = 80.04 Å. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (∼+50°) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2′- endo conformation while the rest, including the bulges, are in the C3′- endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (∼4.4 Å) are significantly shorter than the average P-P distance in the duplex (6.0 Å). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.