J.

Mol. Biol.

(1991)

217, 113-124

Nuclear Magnetic Resonance Studies of the Hammerhead Ribozyme Domain Secondary Structure Formation and Magnesium Ion Dependence Hans A. Heus and Arthur

Pardi?

Department of Chemistry and Biochemistry University of Colorado at Boulder Boulder, CO 80309-0215, U.S.A. (Received 8 June 1990; accepted 21 August

1990)

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide noncleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1 : 1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by twodimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C-G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.

1. Introduction Certain plant viroids and virusoids replicate by a rolling circle mechanism where linear precursors, consisting of multiple repeats of the RNA genome, undergo spontaneous cleavage at specific sites to yield unit length RNA genomes (for a review, see Symons, 1989). Phylogenetic comparisons of these self-cleaving RNAs revealed a consensus secondary structure around the cleavage site that has been termed the hammerhead self-cleaving domain (Symons, 1989; Forster & Symons, 1987a). This hammerhead domain consists of three helices of variable length surrounding a core of conserved nucleotides. Interestingly, these core nucleotides are situated mainly in single stranded regions of the proposed secondary structure of the hammerhead t Author addressed.

to whom

0022-2836/91/010113-12

all correspondence

$03.00/O

should

be

domain (Forster & Symons, 19876). Truncation and mutation experiments showed that fewer than 50 nucleotides are required for full activity of the hammerhead domain (Buzayan et al., 1986; Forster & Symons, 19876), making this system an attractive candidate for physical characterization. Although in vivo the hammerhead RNA cleavage reaction is intramolecular, in vitro studies show that the hammerhead domain can be constructed from two (or even 3) RNA molecules where the cleavage occurs by an intermolecular reaction (Uhlenbeck, 1987; Haseloff & Gerlach, 1988; Koizumi et aE., 1988; Jeffries & Symons, 1989). In these systems the RNAs associate by base-pairing, which then leads to extremely simple rules for the design of highly restriction enzymes. sequence-specific RNA Sequence-specific RNA cleavage activity for hammerhead ribozymes has been demonstrated in vitro (Uhlenbeck, 1987; Haseloff & Gerlach, 1988) and, more recently, in vivo (Cotten & Birnstiel, 113

0 1991 Academic Press Limited

H. A. Heus and A. Pardi

114

1989; Sarver et al., 1990). However, in vitro the cleavage activities of various hammerhead ribozymes on their complementary substrates vary by over a factor of 1000 (Ruffner et al., 1989; Fedor 81 Uhlenbeck, 1990). The differences in activity seem to originate primarily from equilibria between alternate (inactive) conformations for the ribozyme or the substrate, which compete with formation of the hammerhead complex. Quantitative studies by Fedor & Uhlenbeck (1990) have shown that the differences in activities arise from changes in the K, for the reaction with the k,,, values being very similar for a variety of hammerhead RNA enzymes that cleave different target RNA sequences. Non-denaturing gel electrophoresis was used in this study to show that some RNA substrates form stable, inactive, structures that are in equilibrium with active structures. lH nuclear magnetic resonance (n.m.r.t) has also been used in a similar manner to search for structural heterogeneity, and we have recently shown that the hammerhead ribozyme can also form alternate (potentially inactive) structures (Heus et al., 1990). RNAs in biological systems are found to form a variety of structural motifs including stable hairpin (stem-loop) structures, pseudoknots, as well as tertiary interactions such as those found in tRNAs (Saenger, 1984; Pleij et al., 1985; Gutell & Woese, 1990; Cheong et al.: 1990). Since there is relatively little structural data on such RNA systems, it is expected the number and type of these RNA folding motifs will increase significantly as more direct struetura,l data is obtained. So although there is at present a great deal of biological, biochemical and kinetic data on the in vivo and in vitro activities of the hammerhead self-cleaving RNA, there is no direct, structural information on this hammerhead domain. Here we present the first n.m.r. structural data on a hammerhead complex. The systems being studied consist of non-cleavable DNA substrates bound to hammerhead RN$ enzymes. ‘H n.m.r. data are obtained on three similar hammerhead complexes that differ in sequence (and stability) of one of the “recognition” helices. Since a divalent ion, such as magnesium, is required for efficient catalytic activity of the hammerhead ribozyme (Uhlenbeck, 198’7; Symons, 1989), the n.m.r. structural data were also obtained as a function of the Mg2 + concentration. The n.m.r. dat,a on these hammerhead complexes in the absence and presence of Mg2+ will be discussed in terms of secondary structure formation in the Eiammerhead domain. 2. Materials (a) Preparation

and Methods of the RNA enzymes

The 3 ribozymes were synthesized by in witTo transcription with phage T’7 RNA polymerase using synthetic t Abbreviations used: n.m.r., nuclear magnet,ic resonance; TEAB, triethylammonium bicarbonate; 2D, two-dimensional; NOE, nuclear Overhauser effect; ID, one-dimensional; p.p.m., parts per million.

DNA templates by procedures similar to those previously described (Milligan et al., 1987; Heus et al., 1990). One or more large-scale transcription reactions were performed for each ribozyme. A typical 100 ml transcription reaction contained 200 nM-DNA template, 4 mM of each NTP a.nd 10 mg of T7 R?GA polymerase in 40 rnM-Tris. HCI, 5 mM-dithiothreitol, 1 mnr-spermidine, 25 mM-MgCl,, 601% (v/v) Triton X-100 (pH &l), with incubation at. 37 “C for 4 h. For R8 and Rll the transcription reactions were purified as described by Heus et al. (1990). For the R13 transcription reactions the crude Rh‘A mixture was diluted to 250 ml with H,O and applied to a 30 ml fractogel DEAE column (Supeleo). which was equilibrated with 175 mm-TEAB (pH 8.0). The column was washed with 175 mM-TEAB until the unreacted Pu’TPs and short abortive RKA transcripts were eluted, after which the larger RPU’B transcripts were eluted with 1 M-TEAR (pH 8.0). The RXA elutant (30 ml) was concentrated by 3 cycles of evaporation and dissolution in 5 ml of hrgh pressure liquid chromatography grade methanol. This treatment was found to yield better separation of the RNA products during the gel electrophoresis. It is known that in addition to produ&g the full lengt’h product, in vitro transcription with T7 RKia polymerase also generates some oligomers that have an extra nucleotide or 2 in addition to those complementary to the DKA template [Milligan et al., 1987). Preparative polyacrylamide gel electrophoresis was used to separate the full-length transcript from the other RxAs. Typica,lly 8 to 15 15(& (w/v) polyacrylamide gels (40 cm x 60 cm x 0.3 cm) containing 7 M-urea were run for 10 to 12 h at 800 V. until the bromophenol blue dye marker reached the bottom of the gel. The band corresponding to the full-length transcript was identified by ultraviolet shadowing and was eMed from the gel with 50 rnx-potassium acetate, 200 m&uKG1, IO mw-EDTA. The eluted RPL’A was then concentrated on a 3 ml fractogel DEAE column and desalted as previously described (Heus et al., 1990). All purified RNAs migrated as single bands on denaturing polyacrylamide gels. This procedure gave final yields of 6 to 13 mg of purified RSA enzymes from 100 to 200 ml transcript’ion reactions. (b) Preparation

of the DLVA 02iyormm

The DKA oligomers used in the n.m.r. studies and those used as templates for the T7 RKA polymerase were synthesized on an Applied Biosystems 38OB solid phase DNA synthesizer in 02 to 10 pmol scale reactions. Purification of the oligomers was performed with reverse phase chromatography on a C-18 column by standard methods (McLaughlin & Piel, 19&t), followed by desa,lting using a G25 gel filtration column as previously described (Heus et al., 1990). This procedure yields about 1.5 mg purified DNA/pmol synthesis. (c) n.m.r. spectra The RlCA n.m.r. samples were prepared by dissolving the purified oligomers in 0.60 ml of BOoi, H,OjlO% ‘W,O, 10 mlvr-sodium phosphate, 61 m&r-EDTA @I M-KaCl, pH 7.0). The sampies were heated to 85°C for 2 min and slowly cooled to room temperature over 20 min. The RSA and DP;A concentrations were calculated from absorbantes at 260 nm that were corrected for hypochromieity effects determined from the optical melting curves using extinction coefficients calculated from nearest neighbor values (Fasman, 1979). For the DKA titration experiments, a concentrated DNA stock solution was added directly to the n.m.r. samples and the total volume of

n.m.r. Studies of Hammerhead Ribozyme Domain DNA solution added was approx. 25 ~1. After each step in the titrstion the sample was heated to 85°C and slowly cooled to allow for complex formation. For the magnesium titration studies a concentrated solution of MgCl, was added directly to the n.m.r. sample but no heat-cool step was performed in order to prevent degradation of the RNA in magnesium at high temperature. After magnesium titration, the hammerhead RNA enzyme-DNA substrate complexes were dialyzed against the appropriate buffer using a Centricon 3 ultrafiltration apparatus to fix the free concentration of magnesium ion more precisely in the n.m.r. samples. All n.m.r. spectra were recorded on a Varian VXR-500s n.m.r. spectrometer operating at 499.8 MHz for protons at a temperature of 15( kO2) “C. One-dimensional n.m.r. spectra were recorded using a 1331 binomial water suppression pulse sequence (Hore, 1983) with 8192 complex data points and a 10,000 Hz spectra width. The 2D NOE spectra were recorded in the phase-sensitive absorptive mode with quadrature detection in both dimensions using the hypercomplex method (States et al., 1982). The 2D NOE pulse sequence used a 1331 water suppression pulse as the acquisition pulse and a 200 ms mixing time. The acquisition parameters were 128 scans and and 4096 complex data points in the t, dimension; 300 complex free induction decays in t, with recycle

115

delays of 1.4 to 1.5s. The 2D n.m.r. spectra were transferred to a Sun 4/260 computer and processed using the FTNMR program (Hare Research, Inc.). The spectrum was apodized by a 65” shifted sinebell window function in both dimensions and a 5th or 9th order polynomial baseline correction was performed over a limited spectral region in w2 after the first Fourier transform.

3. Results and Discussion (a)

Design of the hammerhead

Figure l(a) shows the consensus structure for the hammerhead RNA domain. The hammerhead domain is made up of a core of 13 phylogenetically conserved nucleotides surrounded by three double helical regions (Symons, 1989). We have divided the hammerhead domain into two pieces as shown in Figure l(b) to (d). In this system the ribozyme will cleave targeted RNAs that contain sequences that are complementary to those in helix I and III. Thus, helices I and III form the recognition helices that account for the sequence-specificity of the ribozyme. Uhlenbeck and coworkers have shown that a substrate of complementary sequence comprising

3’

OH5

“’

U-A1 m fA’t‘T1 C-G

111

-1

\ I

AA

25 GGiX G I I I I CCGG A 1’5 G U II lb) 3’

OH5 ’

“’ U-A1 piJ III m Sdll C-G 0-A-R 10 A c GGl&CC3d, I I I I I I I CCAGCG? ppp U

c

i

1

5’

Sd8

30A A

(a 1 3’

complex

OH’ ’

“’ III

25 AA A dOG

R8

U-AI

m’

Sd13 pJ C-G 10 30-A-T-5 A c GGl&CC3

’ I I I I I I I OH

A

CCAGCGT PPP 5’ UC 3 1 I ” AGlo R13 (d)

Figure 1. (a) Consensus structure of the hammerhead RNA self-cleaving domain (Symons, 1989). The arrow indicates the cleavage site, the 3 double stranded stems are labeled I to III, and a line between the strands indicates base-pair formation. The sequences for the complexes are shown in (b) R8-Sd8, (c) Rll-Sdll and (d) R133Xd13. The ribozyme and substrate nucleotides in each complex are numbered separately starting from the 5’ end of the oligomer. The shaded boxes indicate differences in the sequences for the 3 complexes.

H. A. Heus and A. Pardi

116

deoxyribose sugars, except for a single ribose at the cleavage site, is efficiently clea,ved by the RiYA enzyme (0. C. Uhlenbeck, personal communication). This indicates that the ribozyme is active even if helices I and III are RNA-DNA hybrid duplexes. However, a full DNA substrate is not cleaved by the ribozyme, and thus for these n.m.r. studies we used a completely DNA substrate. This system allows us to study the structure of the hammerhead domain as a non-cleavable substrate bound to the RNA enzyme. Figure l(b) to (d) shows the three hammerhead domains being studied. Each domain consists of a 13 nucleotide DNA substrate that base-pairs with a 34 nucleotide ribozyme. The sequences for the stem II and the GA, tetranucleotide loop were chosen because they form a very stable hairpin, which should help to minimize alternate structures in the ribozyme (Heus et al., 1990). The nucleotides in the recognition helices 1 and III in R8 were chosen to cleave a single site in the Tetrahymena rR?JA intervening sequerme (Kruger et al., 1982). The three ha.mmerhead domains differ mainly in the number of C. G base-pairs for helix III; however, in the R13-Sd13 hammerhead domain, we have also substituted a 6:. C base-pair in helix I with a G. T

base-pair. As will be discussed below, the sequence changes for helix III in the Rll and R13 ribozymes were designed to help to stabilize the ribozymesubstrate interaction. (b) dmino

proton

spectra

as a function

oj the hammerhead

complex

of substrate concentration

The n.m.r. spect’ra of each complex were run a,t various ribozyme to substrate ratios. Figure 2 shows the imino proton spectrum of a titration of the R,8 ribozyme with the Sd8 substrate. The proton spectrum between 12 and 15 p.p.m. is used to monitor base-pair formation, since this is the region where the U, T or G imino protons in a base-pair normally resonate. Since the imino protons can exchange w&h solvent water; an imino proton resonance is only observed in the n.m.r. spectrum if its exchange is slow ( > ms) on the n.m .r. time-scale. As previously discussed the spectrum of the R8 ribozyme alone in Figure 2 has six imino proton resonances corresponding to six kinetically stable base-pairs (Heus et al., 1990). Titration with the DNA substrate leads to shifting of some of the ribozyme imino proton resonances as well as the appearance of new reson-

DNA : RNA !:I

0.75:

I

03:l

0.25 : I

0:i

Figure 2. Imino proton spectra for the titration of’ the I38 ribozyme with substrate

to ribozyme

is given

in the Figure.

the Sd8 DKA

substrate.

The ratio

of

n.m.r. Studies of Hammerhead Ribozyme Domain antes in the spectrum. As will be discussed below, these new resonances arise from the formation of base-pairs in helices I and III. At the end point of the titration in the RS-Sd8 complex there are a total of at least 11 imino proton resonances between 12.0 and 14.5 p.p.m, indicating the complex contains at least this many imino protons involved in hydrogen bonding base-pair interactions. The titration of the ribozyme with the DNA substrate can also be used to monitor the stoichiometry of complex formation. For example, the resonance at 10.54 p.p.m. in Figure 2 corresponds to the int.ensity of a single imino proton in the free ribozyme. Upon titration with substrate the intensity of the imino proton resonance at 1419p.p.m. can be used to monitor the formation of the complex where the ratio of the intensity of this resonance to the peak at 10.54 p.p.m. indicates the percentage of complex formed. At a 1 : 1 ratio of ribozyme to substrate (based on concentrations measured from ultraviolet absorption) there is also a 1 : 1 ratio of the intensities of these peaks. Titration experiments were performed on the Rll-Sdll and R13-Sd13 complexes (data not shown), which also show a 1 : 1 stoichiometry for these complexes. (c) Distinguishing

between G and U/T imino protons

2D NOE spectra in 90% H,O were used to assign the imino proton resonances in the three hammerhead domains. The first step in the assignment procedure is identification of the number of imino protons arising from A. U/A. T and G. C base-pairs.

117

The U or T imino protons in a standard Watson-Crick base-pair generally resonate downfield of the G imino protons and thus some indication of base-pair type can be obtained from the chemical shifts. A more useful criteria for of NOE distinguishing base-pair type is the pattern observed in the 2D NOE spectrum. In an A. T/A. U base-pair the pyrimidine imino proton is within 2.8 A (1 A = 0.1 nm) of the aromatic A2 proton, leading to a single, strong cross-peak in the imino proton-aromatic proton region of the 2D spectrum. However, for a G. C base-pair one generally observes two strong cross-peaks in this region of the 2D spectrum. In this case the cross-peaks arise from interactions involving a G imino proton and two exocyclic C amino protons. The imino proton is within 2.6 A of the hydrogen bonded exocyclic C amino proton, thus accounting for one strong NOE cross-peak. The second NOE cross-peak arises from chemical exchange of the hydrogen bonded and the non-hydrogen bonded amino protons by rotation about the C-N bond. Although this chemical exchange is slow ( > ms) on the n.m.r. time scale, the similar sizes for both imino proton-amino proton cross-peaks (see Fig. 3) indicates that the lifetime for this rotation is less than the mixing time (200 ms) in the 2D NOE spectra. Figure 3 shows the imino proton-aromatic proton region of the 2D NOE spectra of the R8-Sd8 and R13-Sd13 hammerhead complexes. Analysis of the chemical shifts and the patterns of NOE cross-peaks leads to the identification of a minimum of nine, nine and ten G. C base-pairs in R8-Sd8, Rll-Sdll

6.5 Non-hydrogen bonded C amino protons

6.5 Non-hydrogen bonded C amino protons

7.0

8.0 Hydrogen bonded C amino protons

145

14.0

8.0

Hydrogen bonded C amino protons

13.5 w2 (f3.p.m.)

13.0

12.5

14.0

135 130 w2 ( p.p.m.)

lmino protons

lmino protons

(a)

(b)

12.5

Figure 3. (a) Contour plot of the imino proton to aromatic proton region of the 2D NOE spectrum of the R8-Sd8 complex in H,O. The ID spectrum of the imino proton region is plotted across the center of the contour plot. The hydrogen bonding and non-hydrogen bonding C amino protons are highlighted and the circle indicates an interbase-pair sequential NOE (see the text). (b) An analogous plot for the R13-Sd13 complex.

118

-_._- ..-...-..

and R13-Sd13, respectively, as well as a single A.U/A+T base-pair in RS-Sd8 and RI 1-Sdll and two A.U/A.T base-pairs in R13-Sd13. Although identification of the number and type of base-pair is useful in determining the extent of secondary structure formation, assignment of imino proton resonances to specific base-pairs in the sequence is required to determine which base pairs and/or helices are formed in the complex. (d) Assignment

of the imino proton resonances for helix I in the hammerhead complexes

Sequential resonance assignment of the imino protons is made by observation of interbase pair NOES in the 2D NOE spectra in H,O. Assignment of the imino protons in helix I in RS-Sd8 was determined from the pattern of sequential imino proton-imino proton NOE cross-peaks illustrated in Figure 4(a). The continuous lines in the Figure trace a connectivity corresponding to a GGG(U/T)G sequence. This pattern of imino proton connectivities does not determine the orientation (3’ or 5’) of the sequence, or whether each connectivity involves an intra or cross-strand interaction. However, inspection of the primary structure for R8-Sd8 in

Figure 1(b) shows that the NOE connectivity pattern in Figure 4(a) sorresponds to the ribozyme 5’ GCGAC 3’ sequence for the non-termina,l basepairs in helix I. The imino protons on the terminal base pairs in helix I are not observed in t,he R8-Sd8 complex due to fast exchange (on the n.m.r. time scale) with solvent H,O indicating that these basepairs are kinetically labile. In addition to the intrabase pair imino protonaromatic proton NOES shown in Figure 3(a), a number of interbase pair NOES are also observed that arise from interactions between protons on neighboring base-pairs. For example, the NOE cross-peak circled in Figure 3(a) corresponds to an interbase pair NOE between the A2 proton in t’he A5. T9 base-pair to t,he neighboring G4 imino proton in the R8-Sd8 hammerhead complex. The sizes for the cross-peaks from interbase pair NOES are much smaller than those for intrabase pair NOES due to the longer distances separat,ing protons on neighboring base-pairs. These interbase pair NOES help to confirm the assignments, and the Chemical shifts for the imino protons in helix P in R8-Sd8 are given in Table 1. To further confirm the assignments for helix I we replaced a G. C base-pair in the RS-Sd8 hammer-

12.4

12.8

13.6

6 813 14.4

14.0

13.6

13.2 wz (P.P.d (0)

12.8

12.4

14.4

14.0

13.6

13.2

12.8

12.4

w2 (p.p.m.) (b)

Figure 4. (a) Contour plot of the imino proton-imino proton region of the ZD KOF, spectrum of the R8-Sd8 compiex described in Fig. 3(a). The 1D spectrum of the imino proton region is plotted across the top of the contour plot. The assignments for the imino proton resonances are given in the ID spectrum with the numbers for protons on the ribozyme being boxed. The connectivities used to make the sequential resonance assignments for helix I are illustrated by the continuous lines below the diagonal. The connectivities used to make the sequential assignments for helix II are illustrated by the broken lines above the diagonal. (b) An analogous plot is shown for the R13-Sd13 complex. A larger spectral region is plotted here in order t,o show the G. T base-pair in helix I (see the text).

n.m.r.

119

Studies of Hammerhead Riboxyme Domain Table 1

Chemical

shifts

(in

p.p.m.)

of the imino

proton

resonances

of the hammerhead

complexes at 15°C Base-pair A. Helix I G2-Cl2 C3-Gll G4-ClO/TlO A5-T9 C6-G8 B. Helix II G15-C26 G16-C25 Cll-G24 C18-G23 0. Helix 111 D. Other G19 ;

R8

R8Sd8

Rll

13.20 12.72 12.40 1419 13.05

RllSdll

RI3

1314 12.69 1241 1417 12-99

R13Sd13 1314 1276 1157/1919 14.24 12.96

12.55 13.11 13.20 12.50

1258 1313 13.23 1250

1255 1309 1319 1248

1262 1311 13.19 12.48 1354 (U32-A3)

12.59 13.13 13.22 12.49

12.64 13.12 13.21 12.48

1054

10.54

10.54

10.52

10.56

1052

991

1036 $97 W;;@C)S

987

10.43 1001 ‘1290 (GC)

991

1008 10.44 d13.25 (AU/T) ‘12.96 (GC) ‘12.83 (GC)

The chemical shifts are referenced to the residual water signal, which resonates at 4.90 p,p.m. at this temperature. -t a to f Indicate unassigned base-paired imino proton resonances. $ Assignment to base-pair type is given in parentheses.

head complex with a G. T base wobble base-pair in the R13-Sd13 complex. Figure 4(b) shows the imino proton to imino proton region of the 2D NOE spectrum for the latter complex. The imino proton resonance at 12.4p.p.m. assigned to the G4-Cl0 base-pair in the R8-Sd8 ribozyme spectrum disappears in the R13-Sd13 spectrum and two new imino proton resonances appear at 10.19 and 11.57 p.p.m. A G. T wobble base-pair has two imino protons, each of which are hydrogen bonded to a carbonyl oxygen causing these protons to resonate between 95 and 12 p.p.m. Since there is a short distance ( < 2.6 A) between the two imino protons in a G. T base-pair, the strong NOE between proton resonances at 11.57 and 1@19 p.p.m. in Figure 4(b) assigns these signals to the G. T base-pair. The continuous lines in the lower part of Figure 4(b) trace out the sequential imino proton connectivities for helix I in the R13-Sd13 complex. No connectivity is observed from either of the G. T wobble imino protons to the neighboring Gil imino proton. However, all other sequential connectivities for the non-terminal base-pairs in this helix are observed in this complex. The assignments for helix I in R13-Sd13 are again confirmed in some cases by interbase pair NOES. For example, Figure 3(b) highlights an NOE from the A2 proton for A5 on the ribozyme to the imino proton on G8 in the substrate. The chemical shifts for the imino protons in helix I in the R13-Sd13 hammerhead complex are very similar to those observed in the R8-Sd8 complex and are listed in Table 1. An analogous procedure was used to make assignments of the imino protons in helix I for the Rll-Sdll complex

(data not shown) Table 1.

and these results

are also given

in

(e) Assignment of the imino proton resonances in helix II The sequential assignment procedure was used to assign the four G. C base-pairs in helix II in the three hammerhead complexes. The stem II helix and loop, as well as the nucleotides flanking this helix, have exactly the same sequence in all three hammerhead complexes. Thus, one would expect the n.m.r. properties for this region of the molecule to be very similar in all three complexes. The presence of these four G-C base-pairs is confirmed by the spectra shown in Figures 3(a) and 4(a), with the latter illustrating the sequential connectivities used to assign the four G. C base-pairs in helix II in the R8-Sd8 complex. These connectivities are also seen in the R13-Sd13 (Fig. 4(b)) and the Rll-Sdll (data not shown) complexes. As discussed above, the procedure used for sequential resonance assignment of the imino protons does not give the orientation of the helix. So although a stretch of four connectivities between G imino protons is seen for the base-pairs in helix II, the 2D NOE data alone do not give the absolute assignments. The final assignments were made by several additional pieces of information. First, upon titration of the R8 ribozyme with the Sd8 substrate the G imino proton resonance at 1255 p.p.m., which is on one end of the stem II helix; shifts more (up to 0.07 p.p.m. in Rll-Sdll) than the imino proton at

120

El. A. Heus and A. ,Pardi

the other end of the helix (at 12.50p.p.m.). The imino proton next to the conserved core nucleotides is more likely to shift upon complex formation and therefore the resonances at 1250 and 12.55p.p.m. are assigned to Cl&G23 and G15-C26, respectively. These assignments are confirmed from independent assignments of a small RNA hairpin that, contains the GA, tetranucleotide loop connected to a stem that has the same sequence as that in the hammerhead ribozymes (H.A.H. & A.P., unpublished results). The resonances for the three G19 imino protons in the GA, loop in each of the three hammerhead complexes were again assigned (see Table 1) by comparison with spectra of the small RNA hairpin. The chemical shifts of this imino proton indicate that it is not involved in a standard base-pairing interaction. (f) Assignment

of the imino proton in helix III

resonances

The major differences in the sequences of the three hammerhead complexes occur in helix III. All three complexes contain the phylogenetically conserved T5. A30 and G4. C3 1 base-pairs, however the R8-Sd8 complex contains no other C. G basepairs, whereas the Rll-Sdll and R13-Sd13 complexes contain one and two additional C. G base-pairs in this helix, respectively. Based on thermodynamic studies of double stranded oligonucleotides, these C.G (instead of AU/AT) basepairs should increase the stability of the helix (Freier et al., 1986). The sequential assignment procedure used to assign the imino protons in helix I and IT could not be used to assign the imino proton resonances in helix III. For example, in R8-Sd8 one would predict a sequential connectivity for helix III of UUTGT. However, the 2D NOE spectra in Figures 3(a) and 4(a) g.ive no indicat’ion for additional U or T imino protons besides those already assigned to the T9.A5 base-pair. Thus, it would appear that the T and U imino protons in helix III are exchanging too rapidly with solvent to be observed as separate resonances. Although at least two additional G imino proton resonances besides those already assigned to helix I and II are observed within the RS-Sd8 complex, it was not possible to assign these resonances to base-pairs using sequential imino specific proton-imino proton NOES. However, comparison of the imino proton spectra of the three complexes in Figure 5 allowed assignment of one A. U basepair in helix IIT in the Rll-Sdll complex. One of the differences in the three spectra is the broad resonance at 1354 p.p.m. in the Rll-Sdll complex that is absent in the other two spectra. As compared to R8-Sd8, RII-Sdll has an additional C. G basepair in helix I, which will tend to stabilize this helix. Therefore, the resonance at 13.54 p.p.m. is from an A’U/A*T base-pair in helix III that is in intermediate exchange. The most likely candidate for

this base-pair in Rl l-Sdll is U32-A3, because in this complex the A. U base-pair is flanked on both sides by C-G base-pairs. This assignment is confirmed by comparison with the spectrum of the R13-Sd13 complex, where this A. U base-pair is substituted for a C. G base-pair, leading to the disappearance of the resonance at 13.54 p.p.m. The ID and 2D spectra of the R13-Sd13 complex show more imino proton resonances between 12 and 13-5 p.p.m. than the other two complexes. For example, analysis of the 2D NOE data in Figure 3(b) indicates that there is an additional A. U/A. T resonance at 13.25 p.p.m. (indicated by a weak cross-peak at 786 p.p.m.) as well as another C.6: base-pair at 12.83 p.p.m. We are unable to uniquely identify these imino protons by sequential connectivities, but the appearance of these resonances is consistent with the formation of helix III and/or base-pairs formed by the conserved core nucleotides. (g) Bdentijication

of other imino

proton resonances

In addition to the assigned imino proton resonances already discussed above, there are a number of other imino proton resonances between 95 and 11-5 p.p.m. in all three complexes that were not assigned (Fig. 5). Imino prot,ons in this region of the spectrum usually arise from non-base-paired, or non-standard base-pair interact,ions. For example: the G19 imino protons in the GA, loop are observed in all three complexes and resonate between 10.52 and 10.54 p.p.m. There are also two imino protons, designated a and a, which appear at very similar positions in t’he three hammerhead complexes (see Table 1). The 01 and p resonances in all three complexes have larger line widths than t,he G19 resonance, indicating t.hat these protons are exchanging more rapidly with solvent water than the G19 imino proton. Kowever, both these unassigned resonances have narrower line widths in the R13-Sd13 complex as compared with the other two hammerhead spectra, indicating tha,t the stabiliza,tion of helix III may slow down exchange for these imino protons. We are unable to a,ssign these resonances to specific imino protons but’ their chemical shift position and exchange properties indicate that they arise from imino protons not, involved in base-pair interactions, or from imino protons involved in non-standard base-pairs. Figure 5 shows that there are some other very broad resonances around 11.2 to 11.4 and 122 to 12.4p.p.m. in the Rll-Sdll and R13-Sd13 complexes. This broadening is again assumed to arise from intermediate exchange for these p-atom, which makes it impossible to observe cross-peaks for these protons in the 2D NOE spectra. Thus, even though these resonances could be arising from additional base-pair interact,ions, such as potential tertiary interact,ions among t’he conserved core of nucleotides, these larger line widths make it impossible to assign any of these imino protons in our spectra, using standard techniques.

n.m.r. Studies

qf Hammerhead

Ribozyme Domain

121

t11~1111,1111~1111~111,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

14.5

13.5

12.5

II.5

IO.5

p.p.m.

Figure 5. Imino proton spectra for the complexes: (a) R8-Sd8, (b) Rll-Sdll and (c) R13-Sd13. The assignments for the imino protons are given above each plot with protons on the ribozyme being boxed. Several unassigned resonances are indicated by letters or Greek symbols.

In the three hammerhead complexes studied here both stem I and stem II have C. G base-pairs on the ends of the helices that open into the conserved core nucleotides. However, the imino proton for the base-pair at the end of stem II (G15. C26) exchanges slowly enough with solvent water to be observed in the n.m.r. spectrum, whereas the imino proton from the base-pair at the end of stem I (C7. G7) excha.nges much faster and therefore is not assigned. This difference could simply arise from the fact that helix II is an RNA-RNA duplex whereas helix I is an RNA-DNA duplex. Another possibility for this difference is stacking of Al4 and/or G26 on the basepair at the end of stem II. Turner and coworkers have shown that RNA double helical regions can be stabilized by stacking of bases on the terminal basepair in a helix and that purines have more favorable stacking interactions than do pyrimidines (Freier et al., 1986). A third possibility for the slower exchange behavior of the base-pair at the end of helix II is the formation of a G27 ’ Al4 base-pair. Since both t.he G and A nueleotides are strictly

conserved in the a precedent for oligomers (Santa G. A base-pair is

hammerhead domain, and there is G ’ A base-pairs in model RNA Lucia et al., 1990), formation of a a distinct possibility.

(h) The effect of ikfg’+ on the

hammerhead complexes There is an absolute requirement for a divalent ion (such as Mg2+) for efficient catalytic activity for the hammerhead ribozyme (Uhlenbeck, 1987; Symons, 1989). Thus, it is important to see if there are significant changes in the structure of the complex upon addition of magnesium. Since proton chemical shifts are extremely sensitive to conformational changes in a molecule, they provide a good monitor of structural perturbations. Figure 6 shows the titration of the RS-Sd8 complex with from 0 to 30 mM of added Mg ‘+ There are major changes in the imino proton region of the spectrum, including broadening of the resonances and appearance of many new resonances. These new resonances could

El. A. Heus and A. Pardi

122

h2+] A -

20

\

111,11,,,111~,1111,IIII,IIII,IIII,IIII,I,,I,,I,,,lllr,

14.5

13.5

12-5

I I.5

IO.5

p.p.m.

Figure 6. Amino proton spectra of t,he magnesium ion titration of the RS-Sd8 complex. The titration was performed by addition of a connent,rated solution of Mg2+ to the n.m.r. sample and therefore the concentrations in the Figure correspond to added (and not free) magnesium ion in mM (see t,he text).

arise from a number of possibilities, including: ( 1) stabilization of base-pairs in helix III, leading to slower exchange of the imino protons in this helix; (2) stabilization of “tertiary” interactions among the core of conserved nucleotides in the hammerhead complex; (3) stabilization of and/or equilibrium between alternate conformations of the hammerhead complex; or (4) aggregation of the hammerhead complex. Given the present data it was not possible to unambiguously distinguish between these possibilities for the IWSd8 complex and instead we have concentrated our analysis on the magnesium ion dependence of the R13-Sd13 complex. The effect of Mg”+ on the n.m.r. spectrum of dhe R13-Sd13 complex is dramatically different from that of R8-Sd8. Figure 7 shows a sample t.hat was titrated with from 0 to 30 mM of added Mg”, and subsequent81y extensively dialyzed against 5 m;M-magnesium chloride. The Figure shows a,comparison of the imino proton spectrum of the R13LSd13 complex in 0 rnM-free Mg” and 5 mhlfree Mg ‘+. Because of the high nucleot,ide coneentr&ions (O-7 to 1.2 mM) in the n.m.r. samples, and therefore the large number of phosphate groups, there are many potential magnesium ion binding sites. Thus, the added concentration of ;Mg’+ may not be a good indicator of the free concentration, which is why the precaution was taken of dialyzing the hammerhead complexes against 5 mllr-free Mg 2+ . Here we see very little effect on the n.m.r. spectrum of the complex in Mg2’, the only differences being some slight shifts and broadening of the resonances. This absence of a substantial magnesium ion concentration effect on the n.m.r. spectrum of R13-Sd13 is rather surprising. Magnesium ion is

Figure 7. The imino proton spectra of the R13-Sd13 complex in (a) 0 rnM and (‘a) 5 mw-free Mg’+. The samples were prepared by dialyzing against buffer with and without Mg*+ (see the text).

n.m.r. Xtudies

qf Hammerhead

known to stabilize nucleic acid structures and, given the fact that a divalent ion such as magnesium is required for efficient catalytic activity of the hammerhead ribozyme, one might expect a significant structural change for the complex upon addition of magnesium ion. The imino proton n.m.r. spectra for R13-Sd13 show very little change upon that there is little or titration with Mg’+, indicating no structural change for this complex. This result is confirmed by 2D NOE experiments in ‘H,O, which are being used to study the base and sugar protons in the hammerhead complex, where again the spectra are very similar in the absence and presence of Mg2+ (H.A.H. & A.P., unpublished results). All these n.m.r. data strongly suggest that even in the absence of magnesium, the R13-Sd13 complex already exists in a conformation that is capable of these n.m.r. binding a divalent ion. In addition, data show that magnesium ion binding does not induce a significant conformational change in this system.

4. Summary and Implications

for Future Studies

Proton n.m.r. spectroscopy has been used to study the secondary structure of three hammerhead RNA complexes consisting of an RNA enzyme bound to a non-cleavable DNA substrate. 2D NOE experiments in Hz0 for all three hammerhead complexes show direct evidence for the formation of stable secondary structure in two of the three double helices (helix I and II) predicted from the consensus model of the hammerhead RNA domain (Symons, 1989). The three complexes differ mainly in their G+C content, and therefore stability, for helix III. A comparative analysis of the spectra for the three hammerhead complexes leads to assignment of a kinetically labile A. U base-pair in the Rl l-Sdl 1 complex, and these results are consistent with the formation of helix III in this complex. However, even the hammerhead complex with the most C. G base-pairs in helix III (R13-Sd13) does not show imino proton-imino proton NOE connectivities for this helix. This lack of sequential connectivities in the R13-Sd13 complex, and the broad imino proton resonance for the U32. A5 base-pair in the Rll-Sdll complex suggests that the imino protons for base-pairs in helix III are kinetically more labile than the base-pairs in the other two helices. The kinetic lability in these model hammerhead complexes may have implications for other systems, including the complete viroid RNAs, where the hammerhead structure can exist in equilibrium with alternate inactive structures (Forster & Symons, 1987a,b). In fact the stability of helix III has previously been found to be important for the equilibrium between single and double hammerhead structures (Sheldon & Symons, 1989), where the stability of helix III modulates the equilibrium between these alternate structures. The hammerhead cleavage reaction normally requires a divalent ion such as magnesium for activity. Therefore the effect of magnesium ion concen-

Ribozyme Domain

123

tration on the structure of the two complexes RS-Sd8 and R13-Sd13 was also monitored by n.m.r. spectroscopy. For the R8-Sd8 complex, which contains the helix III with the lowest predicted stability, there were large effects on the n.m.r. spectrum upon addition of magnesium ion. However, for the hammerhead complex that contained a more stable helix III (R13-Sd13) there was essentially no effect on the imino proton n.m.r. spectrum upon addition of Mg’+. Therefore the structure of this complex does not appear to change upon addition of Mg2+ Since the hammerhead domain is known to have a magnesium binding site, these data are consistent with a model where the binding site already exists in the magnesium-free complex. This suggests that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here. From the n.m.r. data on the hammerhead complexes it was not possible to uniquely identify any additional base-pairing interactions besides those predicted from the secondary structure model for the hammerhead domain. One might expect that some of the nucleotides in the conserved core of the hammerhead would be involved in tertiary basepairing interactions that fold the domain into a catalytically active structure with an Mg2+ binding site. Even though we did observe a number of resonances in the imino proton spectra indicating additional base-pairing interactions in the three hammerhead complexes (see Fig. 5); these resonances were too broad and/or overlapped to be assigned from our data. The standard procedure used for assigning imino proton resonances in nucleic acids relies upon the observation of imino proton-imino proton NOE connectivities. However, it is important to realize that this procedure may not work for assigning tertiary base-pair interactions because there may not be any short imino proton-imino proton distances for such interactions. Thus, other methods will often be needed to assign these structurally important interactions. One procedure that can be used to assist in these assignments involves the incorporation of isotopic labels (“N or 13C) at a limited number of sites in the molecule. The use of such labels will allow for the application of powerful techniques such as isotope editing and/or three-dimensional heteronuclear n.m.r. to assign and generate structures of RNA molecules. We are pursuing this approach to aid in the assignment of tertiary interactions in hammerhead ribozyme domains. We thank Drs 0. C. Uhlenbeck and M. J. Fedor for helpful discussions. This work was supported in part by grants from the Searle Scholars program of the Chicago Community Trust (%X110) and PI’IH GM3580’7. The 500 MHz n.m.r. spectrometer was purchased with partial support from NIH grant RR03283.

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by P. Wright