Energetics of Hydrogen Bond Networks in RNA - American Chemical

Department of Molecular Biology, The Scripps Research Institute, MB-19, 10550 North Torrey Pines Road,. La Jolla, California 92037. ReceiVed January 1...
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Biochemistry 2002, 41, 14095-14102

14095

Articles Energetics of Hydrogen Bond Networks in RNA: Hydrogen Bonds Surrounding G+1 and U42 Are the Major Determinants for the Tertiary Structure Stability of the Hairpin Ribozyme† Dagmar Klostermeier‡ and David P. Millar* Department of Molecular Biology, The Scripps Research Institute, MB-19, 10550 North Torrey Pines Road, La Jolla, California 92037 ReceiVed January 14, 2002; ReVised Manuscript ReceiVed September 9, 2002

ABSTRACT: The hairpin ribozyme, a small catalytic RNA consisting of two helix-loop-helix motifs, serves as a paradigm for RNA folding. In the active conformer, the ribozyme is docked into a compact structure via loop-loop interactions. The crystal structure of the docked hairpin ribozyme shows an intricate network of hydrogen bonding interactions at the docking interface, mediated by the base, sugar, and phosphate groups of U42 and G+1 [Rupert, P. B., and Ferre-D’Amare, A. R. (2001) Nature 410, 780786]. To elucidate the determinants for tertiary structure stability in the hairpin ribozyme, we evaluated the energetic contributions of hydrogen bonds surrounding U42 and G+1 by time-resolved fluorescence resonance energy transfer using modified ribozymes that lack one or more of the individual interactions. Elimination of a single tertiary hydrogen bond consistently resulted in a net destabilization of ∼2 kJ/mol. The results of double- and triple-mutant cycles suggest that individual hydrogen bonds surrounding G+1 or U42 act cooperatively and form extended hydrogen bond networks that stabilize the docked ribozyme. These results demonstrate that RNAs, similar to proteins, can exploit coupled hydrogen bond networks to stabilize the docking of distant structural domains.

With a large number of RNA structures that have been determined, the molecular principles governing the architecture of RNA are beginning to emerge (reviewed in ref 1). Helical regions can stack in an end-to-end fashion, or dock side by side. Interactions between distant parts of the molecules are mediated by common motifs, such as pseudoknots, loop-loop and loop-helix interactions, ribose zippers, and adenosine platforms. The tobacco ringspot virus hairpin ribozyme, a small catalytic RNA motif required for processing satellite RNA replication intermediates (reviewed in ref 2), consists of two helix-loop-helix elements on adjacent arms of a four-way helical junction (4WJ,1 Figure 1A). In the catalytically active conformation, the ribozyme forms a compact, docked structure where the two loops interact (3-6). The crystal structure of the 4WJ hairpin ribozyme in the docked conformation (7) provides the first direct view of the docking interface, revealing a network of interactions between loops A and B. †

This work was supported by NIH Grant GM 58873 (to D.P.M.). * To whom correspondence should be addressed. Phone: (858) 7849870. Fax: (858) 784-9067. E-mail: [email protected]. ‡ Present address: Department of Experimental Physics, University of Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany. 1 Abbreviations: 2WJ and 4WJ, two-way and four-way helical junctions, respectively; Fl, fluorescein; TMR, tetramethylrhodamine; Pur, purine; I, inosine; 2-AP, 2-aminopurine; (tr)FRET, (time-resolved) fluorescence resonance energy transfer; ssFRET, steady-state fluorescence resonance energy transfer.

Three sets of interactions may contribute to the tertiary structure stability of the hairpin ribozyme. First, a ribose zipper motif involving A10, G11, A24, and C25 connects the two loops via four hydrogen bonds. Second, the G+1 base adjacent to the cleavage site points into a pocket lined by A24, C25, G36, and A38 (loop B) and G8, A9, and A10 (loop A). G+1 forms various hydrogen bonds with the bases of G8, C25, and G36. Third, the U42 base from loop B packs into a pocket formed between the minor grooves of the A22‚ U41 and A23‚A40 (loop B) and C-2‚G11 and A-3‚U12 (loop A) base pairs. U42 is engaged in hydrogen bonds with A22, A23, G11, and U12. We recently probed the contributions of individual hydrogen bonds in the ribose zipper to the stability of the docked hairpin ribozyme using time-resolved fluorescence resonance energy transfer (trFRET) (8). Surprisingly, even though biochemical evidence for the ribose zipper interactions has been presented in the context of a minimal twoway junction ribozyme (5, 9-12), elimination of the zipper only leads to a marginal destabilization of the docked fourway junction ribozyme (8). Consequently, the major determinants of the tertiary structure stability in the 4WJ hairpin ribozyme remain unknown. In an effort to understand the general principles of RNA tertiary structure stability, we investigated the energetic contributions of hydrogen bonds in the U42 and G+1 binding pockets by trFRET using modified ribozymes that lack one or more of the individual interactions.

10.1021/bi025551b CCC: $22.00 © 2002 American Chemical Society Published on Web 11/06/2002

14096 Biochemistry, Vol. 41, No. 48, 2002

Klostermeier and Millar

FIGURE 1: Construct and modifications that were used. (A) Ribozyme constructs. Fl and T refer to the fluorescein donor and tetramethylrhodamine acceptor dyes, respectively, separated from the RNA by six-atom linkers. Modifications were introduced into loop A (G8, G11, U12, and G+1) and loop B (A22, A23, and U42) as shown. Pur refers to purine and I to inosine; 2-AP is 2-aminopurine, and 2′-H denotes 2′-deoxy modifications. Note that this construct contains a C instead of a U at position 12. For purposes of consistency, it is referred to as U12 throughout the text. (B) Chemical structures of modified nucleotides. Chemical modifications relative to the wild type are highlighted with boxes. Replacing A with Pur eliminates the exocyclic amino group as a hydrogen bond donor, while substitution with I places a potential hydrogen bond acceptor in the 6 position, and changes the 1 position from a hydrogen bond acceptor to a donor. Substitution of G with Pur eliminates the exocyclic amino group as a hydrogen bond donor and the O6 as a hydrogen bond acceptor. Position 1 is changed from a hydrogen bond donor to an acceptor. I only eliminates the exocyclic amino group, while A lacks the exocyclic amino group and inverts positions 1 and 6 to hydrogen bond acceptor and donor, respectively. 2-AP retains the exocyclic amino group but eliminates O6 as a hydrogen bond acceptor and inverts position 1 to a hydrogen bond acceptor. Substitution of U with C places a potential hydrogen bond donor in the 4 position, and changes the 3 position from a hydrogen bond donor to an acceptor. 2′-Deoxy modifications eliminate the 2′-hydroxyl as a hydrogen bonding partner.

Hydrogen Bond Networks Stabilize RNA Structure EXPERIMENTAL PROCEDURES Preparation of RNA Samples. RNA strands were purchased from Dharmacon (Boulder, CO). The sequences were 5′-FlAAA UAG AGA AGC GAA CCA GAG AAA CAC ACG CA-3′, 5′-(TMR-)UGC GUG GUA CAU UAC CUG GUA CGA GUU GAC-3′, 5′-GUC AAC UCG UGG UGG CUU GC-3′, and 5′-GCA AGC CAC CUC GCdA GUC CUA UUU-3′. The deoxy modification at position -1 prevents cleavage. The following modifications were introduced, either singly or in various combinations: U42C, A23Pur, A23I, A22Pur, G11I, G+1A, G+1Pur, G+1-2-AP, G+1I, and G8I modifications were introduced, along with combinations thereof. The RNA was purified and annealed in 50 mM Tris-HCl (pH 7.5) and 12 mM MgCl2 as described previously (8, 13). Time-ResolVed Fluorescence Measurements and Data Analysis. Picosecond time-resolved donor emission profiles were collected using time-correlated single-photon counting. Pulsed excitation was achieved with the frequency-doubled (475 nm) output of a mode-locked titanium-sapphire laser (pulse width of 2 ps) with an external pulse picker to reduce the repetition rate from 76 to 3.8 MHz. The emission was detected at 530 nm (16 nm bandwidth) under magic angle conditions using a microchannel plate photomultiplier (impulse response width of 35 ps). The decays were collected in 4096 channels with a time increment of 9 ps per channel. For each ribozyme construct, two separate donor decays were recorded and analyzed. The first, obtained with a sample labeled with only the Fl donor, was fitted using a sum of exponential decays to yield the intrinsic donor lifetimes and corresponding decay amplitudes. The decay of the doubly labeled ribozyme-substrate complex was analyzed in terms of two distinct donor-acceptor distance distributions, corresponding to docked and extended conformers as described previously (14). Nonlinear least-squares fitting of the donor decay recovered the mean donor-acceptor distance for each conformer, as well as their fractional populations. The equilibrium constant for docking, Kdock, the free energy difference between docked and extended conformers, ∆Gdock, and the destabilization, ∆∆Gdock, due to a ribozyme modification were calculated as described previously (8). The nonadditive effect of combining two mutations on the stability of the hairpin ribozyme was quantified in terms of the free energy term δ, defined in Figure 4 in the Results and Discussion. δ was calculated as follows (15):

δ ) ∆Gdock(double mutant) - ∆Gdock(mutant1) ∆Gdock(mutant2) + ∆Gdock(wild-type) where positive values reflect a synergistic effect of the two mutations, negative values are due to partial additivity, no additional effect of mutation 2, or an antagonistic effect, and a δ of 0 reflects additivity. RESULTS AND DISCUSSION Hydrogen Bonds in U42 and G+1 Binding Pockets Stabilize the Docked 4WJ Hairpin Ribozyme. The hydrogen bonding networks surrounding U42 and G+1, observed in the crystal structure of the docked hairpin ribozyme (7), are illustrated in panels A and B of Figure 2, respectively. Since these networks connect nucleotides within loop A with those

Biochemistry, Vol. 41, No. 48, 2002 14097 Table 1: Destabilization Energies of Singly Modified Hairpin Ribozymes H-bond(s) removeda

fraction docked

Kdock

∆Gdock (kJ/mol)

∆∆Gdock (kJ/mol)

0

Wild-Type 0.93 ( 0.005 12.9 ( 0.9 -6.4 ( 0.2

G+1A G+1Pur G+1-2-AP G+1I G+1dG G8I

1-3 1-3 1 and 2 3 4 5

G+1 Binding Pocket 0.75 ( 0.05 3.0 ( 0.6 0.81 ( 0.02 4.4 ( 0.5 0.86 ( 0.003 6.2 ( 0.2 0.86 ( 0.03 6.2 ( 1.2 0.85 ( 0.01 5.7 ( 0.4 0.85 ( 0.01 5.5 ( 0.5

-2.7 ( 0.5 -3.7 ( 0.3 -4.5 ( 0.07 -4.5 ( 0.5 -4.3 ( 0.2 -4.2 ( 0.2

3.7 ( 0.5 2.7 ( 0.3 1.8 ( 0.2 1.8 ( 0.5 2.1 ( 0.2 2.1 ( 0.3

U42C A23Pur A23I A22Pur dU12 G11I

8-10 9 8 and 9 7 6 10

U42 Binding Pocket 0.80 ( 0.003 3.9 ( 0.4 0.84 ( 0.03 5.4 ( 0.1 0.73 ( 0.05 2.6 ( 0.5 0.88 ( 0.002 7.2 ( 0.1 0.84 ( 0.003 5.2 ( 0.1 0.87 ( 0.005 6.6 ( 0.3

-3.4 ( 0.2 -4.2 ( 0.04 -2.4 ( 0.5 -4.9 ( 0.05 -4.1 ( 0.04 -4.7 ( 0.1

3.0 ( 0.2 2.2 ( 0.4 3.9 ( 0.3 1.5 ( 0.2 2.2 ( 0.2 1.7 ( 0.2

a

0 ( 0.3

For the numbering scheme, see Figure 2.

within loop B, each of the hydrogen bonds might contribute to the tertiary structure stability of the docked hairpin ribozyme. To quantify the energetic importance of each hydrogen bond, we have introduced into the ribozyme a series of nucleotide analogues (Figure 1B) that remove individual hydrogen bonds (numbered 1-10 in Figure 2), either singly or in various combinations, and quantified the resulting changes in the distribution of docked and extended conformers by means of trFRET analysis. Analysis of the time-resolved fluorescence decay profiles of the fluorescein donor (attached to the end of the loop A domain) in the presence of the tetramethylrhodamine acceptor (attached to the end of the loop B domain) (Figure 3A) yielded the average donor-acceptor distance for the docked and extended ribozyme conformers, as well as the equilibrium fractions of each species. The donor-acceptor distances obtained for the docked conformers were constant within the range of 35-39 Å among all modified ribozymes. When the lengths of the helical arms carrying the fluorophores are taken into account, this range in interfluorophore distances translates into only a slight variation in the interhelical angles by