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Nucleic Acids Research Pages 2196-2201  

Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs
Introduction
Materials And Methods
   Synthesis, crystallization and data collection
   Structure solution and refinement
Results And Discussion
   Overall structure
   Structural features of G·U wobble pairs
   Hydrogen bonding and crystal packing
   Biological implications
Acknowledgements
References

Crystal structure of an RNA duplex r(G<U>G</U>GCGC<U>U</U>CC)<sub>2</sub> with non-adjacent G·U base pairs

Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs

Ke Shi, Markus Wahl and Muttaiya Sundaralingam*

The Ohio State University, Biological Macromolecular Structure Center, Departments of Chemistry and Biochemistry, 012 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210, USA

Received December 31, 1998 Revised and Accepted March 29, 1999

NDB accession no. AR0011

ABSTRACT

The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U and U·G wobble pairs separated by four Watson-Crick base pairs has been determined to 2.5 Å resolution. Crystals belong to the space group R3; a = 33.09 Å,[alpha] = 87.30° with a pseudodyad related duplex in the asymmetric unit. The structure was refined to a final Rwork of 17.5% and Rfree of 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3[prime]-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3[prime] penultimate phosphates are observed. As expected, the wobble pairs are displaced with guanine towards the minor groove and uracil towards the major groove. The largest twist angles (37.70 and 40.57°) are at steps G1·C17/G2·U16 and U7·G11/C8·G10, while the smallest twist angles (28.24 and 27.27°) are at G2·U16/G3·C15 and C6·G12/U7·G11 and conform to the pseudo-dyad symmetry of the duplex. The molecule has two unequal kinks (17 and 11°) at the wobble sites and a third kink at the central G5 site which may be attributed to trans [alpha] (O5[prime]-P), trans [gamma] (C4[prime]-C5[prime]) backbone conformations. The 2[prime]-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water molecules.

INTRODUCTION

RNA molecules play important roles in life processes, from genetic information storage (1,2) to catalysis (3,4). The G·U wobble hypothesis was proposed by Crick in 1966 to account for the degeneracy of the genetic code (5). In RNA, G·U wobble pairs are frequently found and some are highly conserved (6). The G·U wobble pair in the acceptor stem of Escherichia coli tRNAAla has been shown to be a major determinant for the tRNA identity (7,8) and influence the local helical environment (9). It has been shown that G residue in the exon at the 5[prime]-splice site is used to base pair with U in the internal guide sequence of the intron (10,11). These discoveries highlight the biological importance of G·U wobble pairs. The crystal structure of the wobble pair including other mispairs was first determined in a dodecamer r(GGACUUCGGUCC) (12). Since then, several other structures containing G·U pairs with other mispairs have been reported (13,14). In 1996, we designed an RNA nonamer r(GGGCGCUCC) which can form four possible structures: I, two isolated G·U wobble pairs and six Watson-Crick base pairs; II, two isolated C·U mispairs and six Watson-Crick base pairs; III, two isolated U bulges and eight Watson-Crick base pairs; IV, a five-mispair duplex (Scheme 1). The structure turned out to be I, as predicted by RNAdraw (15), which gives an opportunity to discuss the effect of only the G·U wobble pairs on the double helix.


Scheme 1.


Figure 1. (|Fo| - |Fc|) electron density omit map at 2.5[sigma] clearly showing the density for the isolated wobble pairs. (a) G2·U16; (b) U7·G11.

MATERIALS AND METHODS

Synthesis, crystallization and data collection

The RNA nonamer r(GGGCGCUCC) was synthesized by the phosphoramidite/TBDMS protocol using our in-house Applied Biosystem Synthesizer 391. The RNA was precipitated by ethanol in the presence of 2.5 M ammonium acetate at -25°C and purified by ion exchange column on FPLC. The sample was lyophilized and crystallized by the hanging drop vapor diffusion method at room temperature. A droplet containing 1 mM RNA (single strand), 25 mM sodium cacodylate buffer (pH 6.0), 1 mM calcium chloride and 0.5 mM spermine tetrachloride was equilibrated against 500 µl 40% MPD in the reservoir. Crystals appeared in 2 weeks and grew to dimensions of 0.2 × 0.2 × 0.5 mm3. A crystal was mounted in a thin-walled glass capillary and sealed with wax. The X-ray data were collected at room temperature on our R-AXIS IIC imaging plate with graphite monochromated CuK[alpha] beam at 50 kV/100 mA. The crystal-to-detector distance was 140 mm and 2[thetas] angle was set to 0°. Twenty-five oscillation frames with 6° [phis]-scan width yielded 1403 independent reflections at 2.5 Å resolution [F [ge] 2.0[sigma](F)]. The frames were processed using the software version 2.1 supplied by the Molecular Structure Corporation. The crystal was in the rhombohedral space group R3 with unit cell constants a = 33.09 Å, [alpha] = 87.30° with one duplex in the asymmetric unit. The volume per base pair is 1337 Å3.

Structure solution and refinement

The nonamer duplex has a similar unit cell constant to the octamer r(GUAUAUA)dC (16) previously done in this laboratory. Using the octamer model, the rotation-translation search showed that the nonamer molecules were in the same position as the octamer; in other words, the structures are isomorphous. Therefore, the octamer was used as the starting model in the refinement by the X-PLOR program (17). The initial rigid body refinement with 358 reflections, from 10 to 4.0 Å, yielded an R-factor of 0.493. Ten percent of randomly selected reflections were used for R-free calculation. Further rigid body refinement dropped the R-factor to 0.432 using 10-2.5 Å reflections. A positional refinement lowered the R-factor to 0.353. The bases in the model were corrected by calculating several cycles of ¦Fo¦ - ¦Fc¦ omit maps (Fig. 1) and viewed by CHAIN (18). The ¦Fo¦ - ¦Fc¦ omit maps showed the presence of G·U and U·G wobble pairs, hence the structure was represented by Scheme 1. Continuing the positional and individual temperature-factor refinement with the corrected octamer lowered the R-factor to 0.220. Next, simulated annealing by heating to 3000 K and slow cooling to 300 K in steps of 0.5 fs time interval yielded an R-factor of 0.209. The terminal 3[prime] ribo-nucleoside did not have any electron densities, but the 3[prime] phosphate group density was clear. Including the 3[prime] phosphate in the refinement dropped the R-factor to 0.196. A total of 34 water molecules at 2[sigma] cutoff from difference density maps dropped the Rwork/Rfree to the final value of 0.175/0.240. The crystal data and the refinement statistics are summarized in Table 1. The atomic coordinates and structure factors have been deposited with the Nucleic Acid Database (19). The NDB accession number is AR0011.

Table 1. Crystal data and refinement parameters of the RNA nonamer
Duplex r(GGGCGCUCC)2
Space group R3
Cell constant (Å) a = b = 45.68, c = 59.95
[gamma] = 120°
Asymmetric unit 1 duplex
Resolution range (Å) 10.0-2.5
Number of reflections [F > 2[sigma](F)] 1403
Final Rwork/Rfree (%) 17.5/24.0
Volume/bp (Å3) 1337
Parameter file Param_nd.dna (19)
Deviations from ideal geometry
    Bond length (Å) 0.010
    Angle (°) 1.4
    Dihedral angles (°) 6.7
    ‘Improper’ angles (°) 1.4
Final model
    Nucleic acid atoms 346
    Water oxygens 34

RESULTS AND DISCUSSION

Overall structure

The X-ray analysis of the RNA fragment showed that the nonamer adopted the duplex structure I with G·U and U·G base pairs at positions 2 and 7, respectively. The two independent strands in the duplex show an RMSD of 1.02 Å and are related by an approximate 2-fold axis, 178.2° rotation and 0.08 Å translation. The RMSD between the present structure and the model used is 1.50 Å. The 3[prime] overhang cytosines swing out so that the junction base pairs can stack. There are no electron densities for the 3[prime] overhang cytosine nucleosides because they are disordered, but their 3[prime] phosphate groups have clear electron density. The [epsis] angles for the 3[prime]-phosphate groups are larger, 250° on the first strand and 247° on the second strand compared to the average value of 215° for the rest of the nonamer. This increase in the angles of the 3[prime]-phosphates again helps the stacking of the molecules to form infinite helical columns with twist angles of nearly 0° for the junction steps.


Figure 2. A superimposition of the (a) G·U and (b) U·G wobble pairs (filled bonds) in this structure with Watson-Crick G·C and C·G base pairs, respectively (open bonds) in the crystal structure of an A-RNA octamer r(CCCCGGGG). In the wobble pairs, the G is displaced into the minor groove and the U is displaced into the major groove.


Figure 3. The hydration of the wobble pairs. (a) G2·U16; (b) U7·G11. A water molecule bridges the N2 of the guanine and O2 of the uridine and also the uridine 2[prime]-hydroxyl group. In (a), N2 of G2 also shows a hydrogen bond to a 2[prime]-hydroxyl group of a symmetry related duplex C15*.


Figure 4. (a) Stacking of the Watson-Crick G1:C17 base pair with the G2·U16 wobble pair showing a high twist angle of 37.7°. (b) Stacking of the G2·U16 wobble pair with the G3:C14 Watson-Crick base pair showing a low twist angle of 28.2°. (c) Stacking of the Watson-Crick C6:G12 base pairs and the U7·G11 wobble pair with a low twist angle, 27.27°. (d) Stacking of the U7·G11 wobble pair with the C8:G10 Watson-Crick base pair with a high twist angle, 40.6°.

Helical parameters were calculated with CURVES (20). The duplex is slightly over-wound, 10.3 residues per turn, with an average twist of 35 (SD 4)° and a rise of 2.4 (SD 0.4) Å. All sugars are puckered in the C3[prime]-endo conformation and the glycosyl torsion angles are anti. The backbone torsion angles [alpha], [gamma] are in the preferred g-, g+ conformations except residue G5 which assumes the next frequent t, t conformation. The RNA duplex bends by 14° towards the major groove at the G5 step, probably due to the t, t backbone conformation. The other two bends of 17 and 11° occur at the wobble steps. In Lietzke’s dodecamer r(GGCGCUUGCGUC) (21), in addition to the non-adjacent G·U wobble pairs, there are two U·U mispairs. The molecule is bent by ~14° at each wobble site and bent by 24 and 21° at the central U·U mispairs. The intrastrand P-P distances in the bending regions are 6.3, 6.1 and 6.0 Å, respectively, compared to the average value in this structure of 5.6 Å. Similar longer intrastrand P-P distances are also found in the RNA 16mer (22) and the tandem U·G/G·U structure (23). The starting model used has only Watson-Crick base pairs and shows two bends 14 and 6° (16) which may be attributed to t, t conformation in the vicinity of the base pair steps at the same position of the wobble pairs. Thus the bending in the duplex can either be caused by the wobble pairs or by the sugar-phosphate backbone.

Structural features of G·U wobble pairs

The penultimate G·U wobble pairs are sandwiched by the end Watson-Crick base pairs and the middle four Watson-Crick base pairs. Each G·U pair is linked by two hydrogen bonds (Fig. 2) and further stabilized by hydrogen bonding to water molecules (Fig. 3). Particularly the minor groove water molecule hydrogen bonds to the G base amino group and O2 of U and also bridges the O2[prime] hydroxyl group of U. This feature is also seen in other G·U pairs (12,22). However, in DNA, G·T pairs lack the O2[prime] hydroxyl group and the bridge to the O2[prime] hydroxyl cannot occur, but the water-mediated base-base hydrogen bonds could still occur. The above hydrogen bonds may be the reason why the G·U base pairs in RNA are more stable than the G·T base pairs in DNA. Also, these hydrogen bonds may explain why the G·U wobble pairs are nearly as stable as the A·U Watson-Crick pair. In order to estimate the effect of the G·U wobble pairs on the helix, (G1-G2-G3)·(C15-U16-C17) and (C6-U7-C8)·(C10-U11-C12) were superimposed on similar base pairs of the octamer r(CCCCGGGG)2 (24). As expected, the uracil residue moved into the major groove (0.75 Å) and guanine moved into the minor groove by half the distance (0.35 Å) (Fig. 2). The G·U wobble pair is non-symmetrical (Fig. 3), the [lambda] angles at the guanines are much smaller (42.3 and 38.7°) than those at uracils (66.4 and 68.7°). N2 of G2 in the wobble pair is more accessible; in fact, a hydrogen bond between the amino group and the 2[prime]-hydroxyl group of symmetry related C15* is formed in the minor groove. This kind of intermolecular interaction has been shown in the group I intron, where the G·U wobble pair provides the recognition site for the substrate (25). The twist angles between adjacent base pairs G1·C17/G2·U16 and U7·G11/C8·G10 are 37.70 and 40.57° (Table 2), while the twist angles between G2·U16/G3·C15 and C6·G12/U7·G11 are 28.24 and 27.27°, respectively, compared with the average values of 31.6° for the Watson-Crick base pairs (Fig. 4). Similar twist angles has been observed in Lietski’s structure (21). The G·U wobble pairs also display larger propeller twist angles (-18.7 and -20.2°) than that of the Watson-Crick G·C base pairs (average -14.5°).


Figure 5. (a) View of the crystal packing in the unit cell perpendicular to the c axis. (b) View of the crystal packing in the hexagonal unit cell down the c axis.

Table 2. Helical parameters of r(GGGCGCUCC)2
  Twist (°) Rise (Å) Tilt (°) Roll (°) Propeller twist (°)
G:C         -10.85
  37.70 3.25 -1.14 10.34
G:U         -18.75
  28.24 3.30 -2.33 12.63
G:C         -16.20
  31.16 3.27 3.61 14.00  
C:G         -17.59
  30.93 3.01 0.16 15.02
G:C         -15.67
  30.89 3.34 -2.39 5.99
C:G         -13.62
  27.27 3.43 0.18 5.95
U:G         -20.18
  40.57 3.48 3.42 3.31
C:G         -12.64

Hydrogen bonding and crystal packing

The 2[prime]-hydroxyl groups in the C3[prime]-endo puckered sugars are in the axial orientation (26). They project into the minor groove and are available to form hydrogen bonds to the adjacent duplexes directly or through water molecules. Figure 5a shows the parallel packing of RNA helical columns in the crystal lattice. In the contact region (Fig. 5b), the backbones of symmetry related molecules are close together and form direct hydrogen bonds O2[prime]-O2[prime], O2[prime]-O2P and O2[prime]-N2. Four direct contacts are observed: O2[prime] of G5 with O2P of G3* (2.6 Å), O2[prime] of C15 with N2 of G2* (3.4 Å), O2[prime] of C16 with O2P of U15* (2.6 Å) and O2[prime] of C4 with O2[prime] of C4* (3.2 Å). A total of 34 ordered solvent molecules were located and most are in the first coordination sphere; eight water molecules in the deep groove, 12 in the shallow groove and 10 along the sugar-phosphate backbone. The deep groove water molecules are concentrated around the G·U wobble pairs and the junctions involving the terminal bases. Four water molecules on the 3-fold axis, the first three separated by a distance of 4.19 and 4.05 Å and the fourth by a distance of 9.02 Å, make direct or water-mediated contacts with the RNA. The first water molecule 202 on the 3-fold axis interacts with O2[prime]s of symmetry related U16[prime]s (Fig. 6a). The second water molecule 201 forms hydrogen bonds to the water molecules 112 (including the symmetry-related molecules) which then bridge the water molecules 101 linked to the wobble pairs (Fig. 6b). The third water molecule 205 is bridged by symmetry related water molecules 102, with somewhat larger distances of 3.91 Å, which form hydrogen bonds to symmetry related O2[prime] of G3[prime]s (Fig. 6c). G5 phosphate group is in the t, t conformation and the O2P hydrogen bonds to the fourth water molecule 203 on the 3-fold axis (Fig. 6d).

   a - b
   c - d

Figure 6. Water molecules on the 3-fold axis bridge the neighboring duplexes. (a) Water 202 interacts with O2[prime] of U16 and its symmetry related counter parts. (b) Water 201 interacts with the symmetry related water molecules 112 and 101. Water 101 also interacts with the G2·U16 wobble pair. (c) Water 205 on the 3-fold axis hydrogen bonds with water molecule 102 which in turn interacts with O2[prime] of the symmetry related RNAs. (d) Water 203 hydrogen bonds with anionic oxygen O2P of the phosphate of G5.


Biological implications

Our sequence is self-complementary with the triplet base-paired double helix (GGG/CUC) at both ends. This is found in the acceptor arm of E.coli tRNAAla. The G·U wobble pair is the recognition site for this tRNA and can be changed to other mispairs, e.g. A+·C without affecting the identity of the tRNAAla (22). The tRNAAla identity will be changed if the G·U wobble pair is mutated to a G·C Watson-Crick base pair (27). A+·C does not have the N2 group as in G·U which is also used as the recognition site. It was proposed, therefore, that the conformation changes of the G·U wobble are responsible for the recognition (28). In fact, the chemical shift changes in the NMR spectra (29) of a G·U mispair in the P1 helix and the acceptor arm of tRNAAla (9) have been interpreted as due to the influence of the local helical environment by the G·U pair. In the present structure, we observe that the G·U wobble pairs show distinguishing twist angles, larger propeller twists than that of G·C pairs in this structure. These deviations from the normal Watson-Crick base pair may fulfill the recognition requirement for the proteins.

ACKNOWLEDGEMENTS

We gratefully thank the NIH grant GM-17378 and an Ohio Regents Eminent Scholar Endowment for supporting this work. We also acknowledge the partial support for the purchase of an R-axis IIc imaging plate by the Ohio Regents Hayes Investment Fund and the Ohio Regents Eminent Scholar Award to M.S.

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*To whom correspondence should be addressed. Tel: +1 614 292 2925; Fax: +1 614 292 2524; Email: sundaral@chemistry.ohio-state.edu

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