1.76 Å Structure of a Pyrimidine Start Alternating A-RNA Hexamer r(CGUAC)dG

Abstract

The crystal structure of the alternating RNA r(CGUAC)dG with a 3 H -terminal deoxy G residue has been determined at 1.76 A Ê resolution. The crystal belongs to the orthorhombic space group C222 1 , unitcell dimensions a = 29.53, b = 44.61 and c = 94.18 A Ê , with two independent duplexes (I and II) per asymmetric unit. The structure was solved by the molecularreplacement method. The ®nal R factor was 18.8% using 4757 re¯ections in the resolution range 8.0±1.76 A Ê . The model contains a total of 496 atoms and 85 solvent molecules. The two duplexes form the repeating unit and stack in the usual head-to-tail (5 H ,3 H /5 H ,3 H ) fashion into a pseudocontinuous helical column. Almost all of the 2 Hhydroxyl groups are engaged in the three modes of water-mediated interactions to the base N3/O2 atoms, the sugar O4 H atoms and the backbone phosphates. Thus, the 2 H -hydroxyl group of RNA is probably contributing to the stability of the duplexes.

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures

“…This analysis produces a graph of 1/T M versus ln [Mg 2ϩ ] that is sigmoidal+ Figure 1D shows the 1/T M versus ln [Mg 2ϩ ] data fitted to the second model+ The data fit the sigmoidal curve better than the linear fit above+ Therefore, it appears as though magnesium ions bind to the three duplexes in a nonspecific manner+ The values for the Mg 2ϩ binding constants are presented in Figure 2 displays the direct relationship between size of the RNA oligomer and the magnesium ion binding constants to the duplex and single-stranded RNAs+ As observed with the ⌬n values, there is an inverse relationship between the K f /K u ratio and the length of the oligomer (Table 3)+ A 1+5-2+0-fold increase in binding of Mg 2ϩ ions to RNA hairpin structures was previously found (Laing et al+, 1994 (poly(A)-poly(U); Record et al+, 1976)+ The similarity in values determined here to those previously measured for the homopolymers argues further in favor of a nonspecific interpretation for the Mg 2ϩ ion stabilization+ The structure of the hexamer (59CGUACdG) 2 , as solved by X-ray crystallography (Biswas & Sundaralingam, 1998), was used as a target in a series of BD simulations+ The highest occupancy sites are located in the deep groove near the center of the duplex+ The results are similar for either the 1+2 or 2+2 Å spheres (see Fig+ 4)+ The lower occupancy sites, modeled as green polyhedra in the middle drawing and as space filling green spheres at the right of the figure, line the deep groove of the RNA+ Tandem GA base pairs belong to the commonly observed structural motifs in RNA+ Ribosomal RNAs have a marked preference for the sequence orientation 59-GA/AG, which is, after the tandem of GU pairs, the most prevalent tandem of non-Watson-Crick pairs, whereas 59-AG/GA is never observed (SantaLucia et al+, 1990)+ To investigate the interaction of magnesium ions with non-Watson-Crick base pairs, we examined the effect of magnesium ion concentration on the thermal stability of two oligomers with tandem GA whose structures have been elucidated (SantaLucia & Turner, 1993;Wu & Turner, 1996)+ Depending upon sequence, tandem GA can form two types of pairs, either with a cis WatsonCrick/Watson-Crick configuration (imino), (59GGCAG GCC) 2 or a trans Hoogsteen/sugar edge (sheared) conformation, (59GGCGAGCC) 2 (Figs+ 5 and 6; Leontis & Westhof, 2001)+ The AG non-Watson-Crick pairs in the sequence (59GGCAGGCC) 2 form a structure with the helix underwound to favor intrastrand base stacking+ To accommodate the cis Watson-Crick/Watson-Crick geometry, the deep groove is widened by about 5 Å relative to A-form geometry (Fig+ 6;Wu & Turner, 1996)+ The GA non-Watson-Crick pairs in the sequence (59GGC GAGCC) 2 form a structure with the helix overwound to favor interstrand base stacking+ The geometry of the tandem trans Hoogsteen/sugar edge GA pairs causes a narrowing of the deep groove by about 4 Å relative to A-form geometry (Fig...…”

Predicting RNA pseudoknot folding thermodynamics

“…For helix stems of other lengths, we use the gene 32 mRNA pseudoknot helix as a template to generate the P and C 4 atomic coordinates through the virtual bond torsional angles (η,θ) and the bond angles (β P , β C ) ( 42 ); see Figure 1d . The virtual bond torsional angles (η, θ) in the helix are (170°, 210°) ( 41 ) and the bond angles (β P , β C ) are (105 ± 5°, 95 ± 5°) for a rigid A-RNA helix ( 44 ).…”

Predicting RNA folding thermodynamics with a reduced chain representation model

“…Moreover, we obtain the bond angles (b P and b c ) of the virtual bonds in the rigid double-stranded helix regions from the NDB database in http://ndbserver.rutgers.edu/. Specifically, from the A-RNA helix crystal structure measured by Biswas et al (1998), we find that (b P , b c ) = (105 6 5 , 95 6 5 ). With the torsional angles (Z, y) and the bond angles (b P , b c ), we can generate the coordinates for each strand of the helix.…”

“…The blue and magenta colors denote the bonds in the bulge loop. The P and C 4 coordinates in the helix are from the crystal structure of r(CGUAC)dG sequences (Biswas et al 1998 …”