The RNA from various plant viruses have structures at their 3' termini that are recognized by tRNA processing enzymes (Haenni et al., 1982; Rietveld et al., 1982; Joshi et al., 1983; Guerrier-Takada et al., 1988) even though the secondary structures of these viral RNAs do not resemble the typical tRNA cloverleaf (Florentz et al., 1982; Rietveld et al., 1983 ). Pleij and co-workers proposed that a novel tertiary interaction, a pseudoknot allows turnip yellows mosaic virus (TYMV$) RNA to adopt a three-dimensional structure similar to that of tRNA (Rietveld et al., 1983 (Rietveld et al., , 1984 Pleij et al., 1985 Pleij et al., , 1986 . The exact function of the tRNA-like structures at the 3' ends of the plant viral RNAs is unclear. The tRNA-like structure, and thus the pseudoknot, is required for viral replication in brome mosaic virus RNA (Dreher BE Hall, 1988a) and may be a relic of a primitive telomere (Weiner & Maizels, 1987) . Pseudoknots have been proposed to t Present address: Institut de Biologie Moleculaire et Cellulaire de CNRS, 15, rue Bene Descartes, 67084 Strasbourg, France.
$ Author to whom all correspondence should be addressed.
$ Abbreviations used: TYMV, turnip yellow mosaic virus; n.m.r., nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, 2-dimensional nuclear Overhauser effect spectroscopy; s.w., sweep width; COSY, 2-dimensional correlated spectroscopy; D&F, double-quantum filtered; p.p.m., parts per million; DEP, diethylpyrocarbonate. play a functional role in a number of other RNAs (Moazed & Noller, 1987; James et al., 1988; Brierley et al., 1989; Tang & Draper, 1989 ; for a review, see Schimmel, 1989) . A pseudoknot occurs, as shown in Figure 1 , when nucleotides within a loop pair with nucleotides outside the loop, forming a second stem (Studnicka et al., 1978 ). An idealized model for pseudoknot structure was proposed on the basis of biochemical data and structural analogy to tRNA (Pleij et al., 1985; Dumas et al., 1987) . The stem regions stack coaxially in an undistorted A-form geometry. The two loop regions in the pseudoknot are inequivalent; loop 1 crosses the deep major groove of stem 2 and loop 2 crosses the shallow and wide minor groove of stem 1. Chemical and enzymatic mapping experiments, mutational analysis and the functional similarities of the viral RNAs to normal tRNA are consistent with this model (Rietveld et al., 1982; Joshi et al., 1983; van Belkum et al., 1988; Dreher t Hall, 1988b ). An oligonucleotide of 19 nucleotides was shown to form a pseudoknot using nuclease mapping and absorbance melting studies (Puglisi et al., 1988) . However, no structural data on the three-dimensional conformation of a pseudoknot have been presented.
Nuclear magnetic resonance (n.m.r.) provides the most detailed structural information on nucleic acids in solution. Most n.m.r. studies of RNA have concentrated on the exchangeable, base-pairing protons of large biochemically isolated molecules. The structures of tRNAPh' (76 nucleotides) (Reid, Stem and loop regions in the pseudoknot structure are indicated; stem 1 is black and stem 2 is hatched. & Moore, 1986; Gewirth b Moore, 1988) have been studied using imino proton n.m.r. data. The imino protons primarily give information about basepairing.
The plant viral RNAs containing pseudoknots are quite large (>5000 nucleotides) but Pleij and co-workers used RNase H cleavage to yield much smaller fragments (112, 88 and 44 nucleotides) of TYMV RNA (van Belkum et al., 1989) . The n.m.r. spectrum of the imino region of even the smallest fragments is quite complex. Much more detailed data on individual nucleotide and strand conformations can be obtained from the nonexchangeable protons but there have been few such studies of RNA (Clore et al., 1984; Happ et al., 1988; Varani et al., 1989; Chou et al., 1989; Davis et al., 1990; Puglisi et al., 1990) . This paper describes characterization of the pseudoknot structure formed by an oligonucleotide of 26 nucleotides with the sequence shown in Figure 1 . Both biochemical and physical methods were used to determine the structure formed by the oligonucleotide.
Nuclease mapping experiments provided initial evidence that a pseudoknot structure is formed by this sequence. in water permitted the assignment of the imino resonances and demonstrated the coaxial stacking of the two stem regions in the pseudoknot.
Two-dimensional n.m.r. experiments on the non-exchangeable resonances provided estimates of interproton distances and ribose torsion angles, thus yielding a detailed view of the structure of this pseudoknot in solution.
(a) Synthesis of oligmucleotides RNA oligonucleotides were synthesized using bacteriophage T7 RNA polymerase and a synthetic DNA template with a double-stranded promoter region and a single-stranded template region (Milligan et al., 1987) . T7 RNA polymerase was purified as described (Davenloo et al., 1984) . DNA oligonucleotides were synthesized using standard solid-phase phosphoramidite chemistry. Transcription reactions were incubated at 37°C for approximately 2 h in 40 mM-Tris.HCl (pH gl at 37 "(7). 1 mM-spermidine, 5 rnM-dithiothreitol, 001 y0 (v/v) Triton X100, 80 mg polyethylene glycol/ml (relative molecular mass = WOO), 36% mM-MgCl,, 4 mM each nucleoside triphosphate and 5 mM-GMP. The DNA concentration was 63 PM per strand and T7 RNA polymerase was used at 30 units/pi. Typical yields were between 10 to 15 nmol of strand per ml of reaction (2 to 3 A,,, units/ ml). For n.m.r. quantities, reaction volumes were 50 to 75 ml. Reactions were quenched by the addition of EDTA to 50 mM. The reaction mixtures were extracted with phenol, then with chloroform and were precipitated with ethanol. Full-length transcripts were isolated from a denaturing (7 M-urea) 20% (w/v) polyacrylamide gel. Reactions yielded 2 major bands corresponding to the correct length and to transcript with an additional nucleotide at the 3' terminus. Gel electrophoresis allowed good resolution of these products. Resolution was worse when reactions were primed using GMP. Incorporation of GMP at position 1 was about 80% efficient as determined by 2-dimensional thin-layer chromatography analysis of total RNase digests of transcripts primed with GMP and labeled with [a-32P]CTP (Nishimura et aZ.. 1967) .
RNA transcripts were electroeluted from the gel using an Elutrap electro-elution apparatus (Schleicher & Schuell) and then precipitated with ethanol. Final purification involved extensive dialysis (approx. 24 h) using a microdialysis apparatus (Bethesda Research) first versus 5 m&i-EDTA, 10 mivr-sodium phosphate (pH 6.4). then versus water.
The sequence and length were verified by 2-dimensional thin-layer chromatography analysis of products of complete enzymatic hydrolysis of internally labeled or 3' end-labeled transcripts (Kuchino et al., 1980) and by partial digestion of 5' end-labeled transcript with basespecific RNases. A Rio-Rad TSK-125 gel-filtration column was used to ensure that the structure formed was monomeric even at high strand concentration. A buffer identical with the n.m.r. buffer was used and retention time was compared with the retention times of RNA molecules of known structure and size.
Enzymatic and chemical structure mapping experiments were performed as described in the following paper .
(b) Deuteriation of nucleotides GTP and GMP were deuteriated at the 8 position of guanine by incubating in *H,O at 50°C for 24 h (Benevides et al., 1984) . UTP was deuteriated at the 5 position of uridine using a modification of the method of Hayatsu (Hayatsu et al., 1970; Wataya & Hayatsu, 1972; Brush et al., 1988) . A stock solution of (NH,),SOa (25~) was lyophilized twice from 'H,O; 120mg UTP was dissolved in 1.0 ml of 25 M (N*H,),SO, and adjusted to pH 80 using NaO*H. The UTP was incubated for 28 h at 50°C. The reaction was monitored by the disappearance of the H5 resonance and the collapse of the H6 doublet into a singlet in the n.m.r. spectrum. Incubation at higher temperatures or higher pH resulted in hydrolysis to UDP and UMP. The majority of the product was not the d*UTP, but the adduct of bisulfite to UTP (Wataya & Hayatsu. 1972 Concentrations of n.m.r. samples were 1-O to 1.5 mw-strand. All n.m.r. spectra are referenced relative to trimethylsilylpropionate (TSP). n.m.r. spectra of exchangeable protons were acquired using a I-3-3-1 pulse sequence (Hore, 1983) . The carrier was placed on the water resonance with a delay time of 125 pa and a spectral width of 15,500 Hz. One-dimensional NOE were measured using decoupler irradiation with a 1-3-3-1 pulse sequece. The irradiation time was 600 ma. All l-dimensional NOE measurements were done at 4.O"C. NOE difference spectra were obtained by interleaving on and off-resonance irradiation. Typically, 4000 scans were taken to achieve good signal to noise. Double-precision acquisition allowed the use of high (16 bit) digitizer resolution. Decoupler spillover was determined by irradiating at variable offsets from a peak. Spillover was estimated at 100 Hz at the decoupler power used. Difference spectra were processed using 5 Hz line broadening.
NOESY spectra were obtained using the standard pulse sequence and phasing cycling for pure phase spectra (Bodenhausen et al., 1984) . Data sets were 2006 in t, with a sweep-width (s.w.) of 44w28 Hz. A total of 450 to 500 t, values were collected with 128 scans per t,. A relaxation delay of 1.8 to 2.0 s was used. Total acquisition time was approx. 30 h. NOESY spectra were obtained at 400 ma for assignment of the spectrum and 150 ms for distance determination.
NOESY spectra at short ( < 150 ma) mixing time had poor signal-to-noise.
All NOESY spectra were measured at 23°C. Spectra were processed using a skewed sine bell function with a 30" phase shift and a skew factor of 67.
Interproton distances were approximated from NOE cross-peak volumes at short mixing times. The NOE intensities of the fixed CH6CH5 distance (245 A; 1 A = 61 nm) was used as a reference. These intensities varied by only 10 to 20% among the 6 cytosine residues. Volume integrals of resolved cross peaks were measured using an ellipse-based method in FTNMR (D. Hare, Infinity Systems). The mixing time of 150 ma was adequate for estimating intermediate range distances ( > 25 A). Phase-sensitive COSY and double-quantum-filtered COSY (DQF-COSY) spectra were obtained using the standard pulse sequences and phase cycling (Piantini et al., 1982; Bodenhausen et al., 1984) . Data sets were 2000 in t, with a S.W. of 446428 Hz; 128 scans were acquired per t,; typical data sets consisted of 450 to 500 t, values. Data were processed using a sine bell with a 30" phase shift in both t, and t,.
Double-quantum (DQ) spectra were obtained using the 90-r/2-180-T/2-90-t,-90-t, pulse sequence with standard phase cycling (Braunschweiler et al., 1983) . The carrier was placed just upfield from the H2'/H3'/H4'/H5'/H5" region of the spectrum. A S.W. of 400 Hz was used, which resulted in some foldover in the H5-H6 region. The delay time (7) was set to 50 ma to maximize excitation of DQ coherences with coupling of approx. 10 Hz in an AX spin system. Data sets were 2000 in t,; 400 t, values with 96 scans per t, were acquired for a total experiment time of 24 h. Data were apodized using a sine bell with a 30" phase shift.
The assignment of the n.m.r. spectrum of an RNA molecule is a more difficult task than the assignment of the equivalent DNA spectrum, and requires modification of the DNA assignment strategy (Hare et aZ., 1983 ). There are 2 major problems: one involves the intrinsic chemical shifts of protons in RNA and the second arises from the structural diversity of RNA molecules. The resonances in a l-dimensional spectrum of DNA are more dispersed than those in a l-dimensional spectrum of RNA. Aromatic and sugar Hl' protons resonate in approximately the same regions in both RNA and DNA. In DNA the H2'/H2" protons resonate far upfield at 2 to 3 p.p.m., whereas H3' protons resonate at 45 to 50 p.p.m.; only the H4' and H5'/H5" resonances overlap at 40 to 4.5 p.p.m. Unfortunately, the H2', H3', H4', H5'/H5" resonances of RNA all overlap in the region from 3.9 to 50 p.p.m.
To assign a non-exchangeable n.m.r. spectrum of RNA, the double-stranded stem regions must first be identified. Data from chemical and enzymatic mapping experiments give a crude view of secondary structure; assignments of exchangeable imino proton resonances help confirm basepaired regions. In an A-form duplex, a connectivity pathway exists from H8/H6 to the Hl' on its own sugar (3.5 A) and to the Hl' on the 5'-neighboring sugar (4.6 A) (Haasnoot et al., 1984) . Although the distance to the 5'.
neighboring
Hl' is quite long, the distance from the aromatic H6/H8 to 5'-neighboring H2' proton is very short (2.0 8) (see Wiithrich (1986) for short distances in an idealized A-form helix). Therefore, spin diffusion from the H2' to the HI' will give rise to a relatively intense 3' (H8/H6) + 5' (Hl') cross peak in the NOESY spectrum at long mixing times.
RNA aromatic protons can only be identified as pyrimidine or purine; in DNA, pyrimidines can be distinguished in NOESY by the thymidine H6 to methyl cross peak. Incorporation of selectively deuteriated nucleotides during RNA synthesis permits the distinction of nucleotide type, by comparison to the spectrum of the nondeuteriated molecule. The AH2 protons are important, since they provide a link to the more readily assigned exchangeable imino protons. An AH2 proton in a doublestranded region is easily identified by an NOE from the A. U base-paired imino proton. The AH2 protons usually have no close distances to non-exchangeable protons in a B-form helix. However, in an A-form helix, the AH2 protons have NOE connectivities in the 3' direction to the Hl' on the same strand, and in the 5' direction to the Hl' on the opposite strand (both distances approx. 4 A). An AH2 misassigned as a purine H8 will lead to an erroneous assignment pathway that shunts across strands. AH2 protons generally have only very weak NOES in the H2'/ H3'/H4'/H$/H5" region. The AH2 protons, once assigned from the imino spectrum, act as a check on the H8/H6 + Hl' assignment pathway. Assignment of nonhelical regions follows the above protocol if the nucleotides are in a stacked, relatively A-form geometry (i.e. the anticodon loop of tRNAPh'); however, assignment pathways are much more difficult to obtain in unstacked regions. This is a serious problem for RNA structure determination by n.m.r., since frequently the non-helical regions of the structure are of primary interest.
The assignment of the H8/H6 protons in an A-form stem can be checked by the strong internucleotide NOE from 3' (H8/H6) ++ 5' (H2') (approx. 2.a). In general, each H8/H6 proton in RNA will have a number of NOES to resonances in the H2', H3', H4', H5'/H5" region between 39 and 50 p.p.m. The NOE to the H2' should be the strongest. This can be confirmed by checking the Hl'/H5 (5.0 to 6.4 p.p.m.) to H2'/H3'/H4'/H5'/H5" (39 to 50 p.p.m.) region of the NOESY. A strong Hl' to H2' (distance 2.5 to 2.7 A) cross peak identifies the H2' on the same nucleotide.
In DNA, this assignment can be confirmed unambiguously by looking for COSY connectivieies between the Hl' and H2'/H2". However, J,,.,, depends strongly on sugar pucker: J,..,, is approximately 8 Hz for C-Xl-e&o sugar pucker and J,..,, is less than or equal to 1 Hz for C-3'-endo sugar pucker (Altona, 1982) . The small J value for C-3'-endo puckers results in no observed H l'-H2' COSY cross peaks in A-form stem regions. Identification of C-2'.endo sugars is obvious, since they do have strong Hl'-HZ' COSY cross peaks. The assignments of H8/H2/H6/H5 base protons and Hl'/H2' sugar protons are the most reliable in a complex RNA spectrum.
Further assignment of the sugar region in DNA is relatively easy since H3' protons resonate separately from H2'/H2" and H4'/H5'/H5" protons. In RNA the situation is more complex. H3' resonances can be identified by intranucleotide H8/H6 * H3' cross peaks (2.8 A in A-form nucleotides) as well as internucleotide 3' (H8/H6) ++ 5' (H3') connectivities (approx.
Hl'-H3' cross peaks can be observed. Intrasugar
Hl'-H4' cross peaks can sometimes be observed for C-3'-endo or C-2'-endo sugars (distance 3.4 A). This cross peak should be very strong for intermediate sugar puckers (i.e. pseudorotation angle P = 9O", distance 2.2 A).
The H2'/H3'/H4'/H5'/H5" region of a DQF-COSY spectrum of an RNA molecule is crowded. This region in principle contains H2' to H3', H3' to H4', H4' to H5'/H5" and H5' to H5" cross peaks. H5' and H5" are usually degenerate, but can be identified by a strong COSY cross peak (J = -15 Hz) when they are non-degenerate.
If the torsion angle y is approximately g+: as found in both A-form and B-form helices (Saenger, 1984) the H4' to H5'! H5" couplings are small (J < 3 Hz). In non-standard conformations (y = g-or trans) the coupling to the H5' or H5" can be quite large (J > 10 Hz). The major cross peaks in this region for RNA are H3'-H4' connectivities, since J,.,. is 8 Hz for C-3'-endo sugars (Altona, 1982) . H2'-H3' cross peaks are much weaker since J,,,. is only 5 to 6 Hz; the intensity is further attenuated by the complex antiphase fine structure of this cross peak arising from the strong passive coupling to H4' and 3' phosphorus. The antiphase nature of COSY cross peaks makes linewidth a critical factor in determining the amount of cancellation of cross peak components. This is a problem with larger RNA molecules; even in the DQF-COSY of the 26 nucleotide molecule, very few HZ&H3 cross peaks are observed, and only a small fraction of H3'-H4' cross peaks are resolved.
A DQ spectrum contains J-coupling information similar to that in a D&F-COSY. ./-coupled cross peaks will occur at the 2 frequencies of the coupled spins in t,, at the sarnfx DQ frequency (vl +v,) in t,. The advantage of DQ spectroscopy is that there is no diagonal, so that connectivities close to the diagonal in a DQF-COSY are easily resolved in a DQ spectrum. The excitation of DQ tram+ tions depends on the DQ preparation period (z), with maximal excitation for t = (25)-l in an AX spin system. For DQ spectra with 7 = 50 ms, H3'-H4' DQ transitions are maximally excited. In practice, I>& spectra can be useful in further assigning sugar spin systems when used in conjunction with the other methods described above. and 60 mM-Na+. At and below 55°C. the oligonucleotide is resistant to single-strandspecific nuclease S, cleavage except in the loop regions of the pseudoknot.
At 4°C. strong cleavage occurs after the second, third and fourth nucleotides in loop 2 (U17, U,, and U19) (Fig. 2) . At 22°C and 37"C, weaker cleavage also occurs in loop 1 and after the other nucleotides in loop 2 (data not, shown).
The molecule is completely resistant to cleavage by RNase T, (specific for single-stranded G) at temperatures at and below 37°C. Nucleotides in loop 2. which bridges the minor groove, are more accessible to single-strand-specific probes than t,hosr in loop 1; this is also observed for the pseudoknot formed by TYMV RNA (van Belkum et al., 1988 . Autoradiograms of gel electropho retie analys of single-strand-specific nuclease S1 and dot tble-s itrandlecific RNase V, digestions of the oligonuclec otide . RNA as 32P-labeled at the 5' end. Lane 1, control, no er izyme; ,ne 2, nuclease S, cleavage at 85°C in ! imix -NaCl, mm-Mes (pH 63) (denaturing condition 4; lane 3, uclease S, cleavage at 4°C in 5 mM-MgCl,, 6( IrnM -NaCl, cleaves the oligonucleotide in the proposed stem regions of the pseudoknot (Fig. 2) . Cleavage is observed after each of the nucleotides in stem 1 although cleavage is weak after G, and C, 5. In stem 2, cleavage occurs following each nucleotide on the 5' side of the stem (Cs through CIZ). Weak cleavage after G,,-, cannot be correlated to the pseudoknot structure, since a hairpin containing only the stem 2 region shows the same sequence-specific pattern of RNase V, cleavage (data not shown). On the 3' side of stem 2, cleavage is observed only after residues G,, and UZ3. Lack of cleavage near the 3' terminus of stem 2 is consistent with RNase V, cleavage of other hairpins and duplexes. RNase V, does not cleave single-stranded control molecules; cleavage after loop nucleotides U7 and U,, is correlated with stacking.
The solvent accessibilities of the three adenosine residues in the oligonucleotide were compared over a range of temperatures using diethyl pyrocarbonate (DEP) (see the following paper; Fig. 2 ). The carboxyethylation reaction at the N-7 position is sensitive to stacking (Peattie & Gilbert, 1980) . Both A,, and A,,, residues in stem 2 of the pseudoknot structure, show the same extent of modification throughout the temperature range from 4°C to 85°C. As expected for nucleotides in duplex regions, A l1 and AX are only slightly reactive at low temperatures. The mid-point of the percentage cleavage versus temperature curve is approximately 65"C, roughly the same as the optically observed melting temperature (see the following paper). Below 45"C, A,, the first nucleotide of loop 1, is about one third as accessible to modification as it is in the denatured oligonucleotide at 85°C. The midpoint of the melting transition of A, is approximately 5 to 10 deg.C lower than that of A,, and A,,. The DEP modification data, like the cleavage patterns obtained using enzymatic probes, support the existence of stem 2. The increased DEP modification of A, as compared with the adenosine residues in stem 2 suggests that this residue is part of a loop region; however, A, also shows a melting transition so it is partially protected from solvent by the folding of the pseudoknot. Loop 1 is proposed to bridge stem 2 across the major groove of the helix.
The n.m.r. spectrum of the exchangeable imino protons in 5 mM-MgCl,, 50 mM-NaCl, 10 mM-sodium phosphate (pH 6.4) at 4°C is shown in Figure 3 . The total of eight base-paired imino resonances is consistent with pseudoknot formation. The two resonances farthest downfield, at 13.92 and 14.35 p.p.m., The resonances below 11.5 p.p.m. are most, likely due to non-hydrogen-bonded imino protons on uridine residues. The imino resonances were assigned using one-dimensional NOE measurements.
An NOE is observed between protons that are less than 45 to 5.0 A apart.
Since the inter-base-pair distance in both A-form and B-form helices is approximately 3.5 to 3.8 A, an NOE is expected from an imino resonance of one base-pair and the imino protons corresponding to the two flanking base-pairs. In addition, uridine imino protons involved in a Watson-Crick A. U base-pair will have a strong NOE to the adenine H2, which is separated by only 1.9 A from the imino proton. The assignments of the imino protons are summarized in Figure 3 (b) and Table 1 . imino resonances 4, 5 and 6 from the core of stem 2 remain. Resonance 4, which corresponds to the terminal C12. Gzz base-pair of stem 2, remains relatively sharp up to 5O"C, whereas the imino resonance from G3. C1 3 pair 3 has completely broadened. The sequential order of broadening of the imino resonances could be due to differences in either the thermodynamic stability or the kinetic behavior of the two stems. Data discussed in the following paper support the latter. The region between 7.0 p.p.m. and 8.6 p.p.m. contains resonances from AH8, GH8, CH6 and UH6 as well as AH2 protons. For the oligonucleotide described here, this region should contain 29 resonances. UH5, CH5 and sugar Hl' protons resonate in the region between 5.0 p.p.m. and 6.2 p.p.m. The spectrum can be significantly simplified in these two regions through selective deuteriation. Deuteriation of pyrimidine residues at the 5 position is experimentally straightforward for either the nucleotides or the DNA or RNA strands (Wataya & Hayatsu, 1972; Brush et aE., 1988) . Since 11 of the 26 nucleotides of the oligonucleotide are uridine residues, UPT deuteriated at the 5 position was incorporated using T7 RNA polymerase. The region between 5.0 p.p.m. and 62 p.p.m. is significantly simplified by the elimination of 11 UH5 resonances. Deuteriation of UH5s also improves the appearance of the 7.0 to 8.6 p.p.m. region of the spectrum because the UH6 resonances are singlets; the presence of deuterium in the H5 position eliminates the splitting of these resonances.
The first step in the assignment of the nonexchangeable spectrum was the identification of uridine and cytidine H6 resonances. The COSY spectrum of the deuteriated oligonucleotide exhibits six cross peaks due to H5-H6 J-coupling (not shown), and allowed the unambiguous identification of the six cytidine H6 and H5 resonances. Figure 5 shows the H8/H6 to Hl'/H5 region of the 400 millisecond mixing time NOESY spectrum. Strong cross peaks arise at the chemical shifts of the CH5-H6 protons in the NOESY spectrum due to the short fixed 2.45 A. This NOESY spectrum was compared with the NOESY spectrum of the non-deuteriated molecule under identical conditions. Strong cross peaks missing in the NOESY spectrum of the deuteriated molecule, due to UH5-H6 interactions? permitted the identification of 6 of 11 UH6 resonances; the other uridine resonances were not sufficiently resolved to allow assignment.
The assignment of the pyrimidine residues aids in the confirmation of the aromatic to HI' assignment paths shown in Figure 5 ; the paths are consistent with both sequence and secondary structure. Connectivity path 1 (Fig. 5(a) ) encompasses nine nucleotides: 3' U-C-R-C-C-R-R/U-U-C 5' (R is a purine). Only one region of sequence is consistent with this assignment path: 3' U16-C15-G,4-C,3-C,2-A~~-G~c-U~-Cs 6. This path spans the two-stem regions of the pseudoknot. Connectivity path 2 ( Fig. 5(b) ) encompasses five nucleotides: 3' R-R-C-U/R-R 5'. This path corresponds to the 3' terminus: 3' G26-A25-C24-U23-G22 5'. Both regions are double-stranded stems on the basis of assignment of the imino spectrum and chemical and enzymatic mapping. One-dimensional NOES from the two A. U imino protons allowed assignment of the resonance at 7.02 p.p.m. as A,,(H2) and that at 7.71 p.p.m. as A,,(H2). Both AH2 positions have NOE connectivities to two Hl' protons. A,,(H2) has NOES to G,,(Hl') and G,,(Hl'); A,,(H2) has NOES to C12(Hl') and &(Hl').
These NOES from AH2 to the S/-neighboring HI' on the same strand and the Hl' proton across strand in the 5' direction are exactly those expected for an A-form helical geometry. Both assignment paths can be confirmed using the 3' H8/H6 * 5' H2' and Hl'-H2' NOE cross peaks (see below).
The final connectivity path ( Fig. 5(b) ) involves the stem region at the 5' terminus. The purine at 7.77 p.p.m. is assigned at G,(H8), since it has a NOE to its own H1'(5*94*p.p.m.), and to the C,(Hl') (5.71 p.p.m.). The C,(H6) (7.93 p.p.m.) was the only unassigned CH6-CH5 cross peak in the COSY spectrum. The C,(H5) (5.96 p.p.m.) has a second weak cross peak at 5.50 p.p.m. due to a small amount of 5' hairpin structure present . The 5'-terminal G I (H8) was identified by incorporation of dsGMP during transcription. Since T7 RNA polymerase preferentially primes synthesis with GMP rather than GTP, the d*G is selectively incorporated at position 1 (Sampson & Uhlenbeck, 1988). G,(H8) (8.24 p,p.m.) has a NOE to its own Hl' (5.90 p.p.m.) and to the C,(Hl'). NOE connectivities are also observed between loop 1 nucleotides ( Fig. 5(b) ). The farthest downfield aromatic resonance at 8.53 p.p.m. is assigned as A, (HS) and the resonance at 821 p.p.m. is A,(H2). These assignments were confirmed by the presence of both these resonances after incorporation of d*GTP (data not shown). The A,(HB) has a strong cross peak to the A,(Hl') at 6.09 p.p.m. The UH6 at 7.74 p.p.m. has a NOE to the A,(Hl'). This proton, assigned as the U,(H6), also has a NOE to the U,(Hl') at 5.78 p.p.m. Further assignments are summarized in Table 2. (ii) Nueleotide conformation: sugar pucker
The individual nucleotide conformations can be determined using both J-coupling and NOE information. Scalar (J) coupling information is used to characterize the sugar conformation as north (approx. C-3'-endo, 1' = 18") or south (approx. C-2'. endo, P = 162"). For smaller RNA molecules, the sugar conformation can be more precisely determined using additional coupling information (i.e. H2'-H3', HY-H4') (Davis et al., 1996) ; this information is limited in the case of the 26 nucleotide molecule studied here. The two families of puckers represent points of minimum energy for the pseudorotation phase angle, P (Olson & Sussman, 1982; Olson, 1982) . Since the rate of interconversion between these two conformers is fast on the n.m.r. time-scale, the value of J,,,, for a nucleotide is an average of J,,,, for north (J,,, < 1 Hz) and south (J,,.,, = 8-O Hz) conformers weighted by the relative population of each. An empirical formula for determining the percentage of each conformer has been described (van den Hoogen, 1988):
(yoN = 114.9-14.5. (J1,.2,) , showing NOES between H8/H6/H2 and H5/Hl' protons. NOES between H8/H6 and Hl' protons are indicated by continuous lines and those between AH2 and Hl' are indicated by dotted lines. Labeled NOE cross peaks indicated a NOE from that nucleotide's aromatic proton to its own Hl' sugar proton. Conditions were: 5 mM-I$$&, 50 mNaC1, 10 mm-sodium phosphate (pH 64) at 23°C. The mixing time was 466 ms. (a) H8/H6 to Hl' NOE connectivity pathway 1, which spans nucleotides Ui6 to Cs. (b) H8/H6 to Hl' NOE connectivity pathway 2 spans G,, to G,, and from G, to C,. Note that there is no NOE between G,,(H8) and G,(Hl'). The broken line indicates the NOES between A, and U, in loop 1.
region of the D&F-COSY is shown in Figure 6 . Certain nucleotides have observable
Hl'-H2' cross peaks. The unresolved cross peaks correspond to Hl'-H2' cross peaks for loop uridine residues. The strong resolved cross peaks labeled in Figure 6 also belong to loop uridine residues; one has been assigned to U16; the other corresponds to an unassigned loop residue. Weaker Hl'-H2' cross peaks are also observed for U,, G,,, A,, and G,,. These cross peaks have J,,,, less than 7 Hz; the smaller coupling results in cancellation of antiphase components in the cross peak, and attenuated intensity (Widmer & Wiithrich, 1987; Bax & Lerner, 1988) . The A,(Hl'-H2') cross peak is very weak and can only be observed in contour plots at very low levels in the COSY. A larger linewidth for these resonances could explain the reduced intensity of this cross peak in relation to the G,, cross peak, which also has a small coupling. The sugar conformations (%N) calculated from these cross peaks using equation (1) are listed in Table 3 . The sugar pucker was estimated as greater than 85% north in the absence of a detectable Hl'-H2' cross peak (i.e. J 1,-2, values less than 2 Hz). Figure 6 . Portion of the double-quantum filtered COSY spectrum of d5U-incorporated oligonucleotide showing cross peaks between sugar Hl' and H2' protons. Cross peaks are only observed for nucleotides with a significant population of S-type sugars; these cross peaks are labeled. The unresolved peaks between 6 and 45 p.p.m. are due to unassigned loop nucleotides.
oligonucleotide.
The method employed here makes use of the known sugar pucker (determined as described above) and the intranucleotide distances (determined from the NOESY). Contour plots that relate both pseudorotation phase angle and glycosidic torsion angle to values of intranucleotide interproton distances were presented by Wiithrich (1986) . If the sugar adopts a north conformation, as do most of the sugars in the stem region, and the value of 1 is anti, then a short H8 or H6 to H3' and a long (approx.
3.8 A) H8 or H6 to H2' distance results.
Assuming a C-3'-endo sugar pucke:
(P = 187, an approximation, since the pseudorotation phase angle is not known precisely, the value of x can be estimated from the H8/H6-H3' distance. The nucleotide conformations determined in this way are listed in Table 3 .
Interproton distances (listed in Table 2  Table 4 Assignments of non-exchangeable protons in 5 m&f-MgCl,, 50 mbf-NaCl, 10 mM-sodium phosphate (pH 6.4) at 23°C give an adequate signal-to-noise ratio. This mixing time will definitely lead to spin diffusion (Keepers & James, 1984; Chazin et al., 1986) . The magnetization transferred by spin diffusion will result in an underestimation of long interproton distances and an overestimation of short distances ( < 2.4 A). Most distances between 2.5 A and 34 A seemed approximately correct, however, as shown by the reasonable values obtained for the relatively short (25 to 2.8 A) Hl'-H2' distance (see below). Nucleotides C, through C15 all adopt north sugar conformations as shown by the lack of an observable Hl'-H2' COSY cross peak for any of these residues. These nucleotides all have intranucleotide proton-proton distances (2.7 to 3.1 A) consistent with an anti value of x. The approximate values of x determined for these nucleotides range between -160" and -175"; the value of x in an A-RNA duplex determined from fiber diffraction is -170" (Saenger, 1984) . U,,, which marks the end of this connectivity path, has an S-type (C-2'-endo) sugar conformation.
The values calculated for both t&e H6-H2' distance (~2.5 A) and H6-H3' distance ( < 2.7 A) are short. No glycosidic torsion angle is consistent with an S-type sugar and both short H6-H2' and H6-H3' distances. The apparent short distance between H6 and H3' is probably due to spin diffusion from the strong H6-H2' connectivity (the distance could be approximately 2 A). The short H6 to H2' distance suggests that this nucleotide has an anti value of 1.
The sugar conformations at the 3' end of the sequence are affected by terminal end fraying. Both GZ6 and A,, have small (<3 Hz) Hl'-H2' coupling. These nucleotides have sugar puckers in fast exchange between N and S-type sugar conformations. GZ6 has an especially large (approx. 40%) population of S-type sugar. The distance deter-J. D. Pug&i et al.
mined by NOE is not a simple average of the distances in the two conformations, but is weighted by the l/r6 dependence towards the shorter distance. For example, Gz6(H8) has short apparent distances to both the HZ' (3.1 L%) and H3' (2.6 8). Nonetheless, these distances are consistent with an anti value of x. A,, has H&H1
(3.4 A) and H&H3 (2.7 A) distances consistent with an anti glycosidic torsion angle.
The population of S-type sugar conformation must be low, since there is not a strong H&H2 NOE. Cz4 and c',, both have pure N-type sugar conformations; the H6-Hl' and H6-H3'
distances give values of x consistent with A-form geometry.
Gz2 mostly adopts a south sugar conformation (approx. 80% S), despite its location in a duplex region (see discussion below). The calculated H8-Hl' distance is short (3.0 A), most likely due to overlap of cross peaks and/or spin diffusion from the strong H8-H2' cross peak. The calculated H8-H3' distance (3.0 A) is short due to the population of N-type sugar present,. G22 has an anti value of x. G, has an N-type pucker. C2 has an N-type sugar conformation; the intranucleotide H6-Hl' distance (3.5 8) is consistent with an anti value of x. G3 also has a pure N-type sugar conformation and both H8-Hl'
(3.4 A) and H8-H3' (2.5 A) are consistent) with an A-form value of x ( -155"). Residue A, may have a non-standard nucleotide conformation. The sugar conformation is mostly N-type. The H8%Hl' distance is quite short (2.8 A), as are both the H8-H2'
(3.0 A) and H8-H3' (3.1 A). Spin diffusion does not seem to be a significant problem with this residue since both the Hl'-H2'
(2.7 8) and HI'-H4' (2.9 A) are consistent with the N conformation. The intranucleotide distances are roughly consistent with a x value in the high anti range (-60" to 0"); however, this nucleotide may be in equilibrium among various conformations.
The sugar conformation of U5 is a mixture of N and S-type puckers. The H6-Hl' distance (3.2 A) is consistent with an anti conformation. The conformation of the remaining loop residues cannot be determined due to spectral overlap and the lack of a clear connectivity path. Many of the loop nucleotides must have some population of S-type sugars, as shown by the number of strong overlapping Hl'-H2' cross peaks in the COSY.
(iv) Strand confomation
The analysis of nucleotide conformations demonstrates that the residues composing the stem regions adopt C-3'-endo sugar puckers with glycosidic torsion angles consistent with A-form helical geometry.
The internucleotide NOES are also consistent with an A-form helical structure in the stem regions. The strong 3' H8/H6 ~15' H2' NOES that are expected for A-form are observed. These internucleotide NOES are difficult to interpret in terms of distances.
The expected 3' H8/H6 ++ 5' H2' distance for A-form is extremely short (2-O A); spin diffusion will cause an overestimation of these Figure 7 . Summary of the n.m.r. data on the oligonucleotide. Each of the nucleotides indicated in large type adopts an anti glycosidic torsion angle. Sugar conformation is indicated by letter type: C-Y'-endo. bold-face type; C-2'.endo, outline; equilibrium between conformations, box. Internucleotide NOES are represented by the thickness of the line connecting 2 nucleotides. Thick lines represent strong 3' H8/H6 tf 5' H2' NOES. consistent with A-form stacking. Thin lines indicate no observed NOE interaction.
No NOE was observed between the non-exchangeable protons of G,, and G,. Figure 7 .
The n.m.r. data described above lead to t.he following conclusions concerning the structure of the pseudoknot.
Residues C, to C,, form a continuous A-form helix. There seems to be little perturbation of the structure along the strand at the junction of stem 1 and stem 2. The NOE connectivity is continuous from Cl2 to C,,: the H5-H5 NOE observed between these two nucleotides at 400 milliseconds mixing time confirms that these two bases are stacked in an A-like fashion. The eight base-pairs form a continuous helix with no break in NOE connectivity between imino resonance 3 and resonance 4 at the junction between the two stem regions. U,, is stacked upon C, 5, continuing the A-form connectivity into loop 2. The non-hydrogen-bonded imino proton of U1, at 10.93 p.p.m. gave a NOE to the (degenerate) imino resonances belonging to base-pairs 1 and 2. It appears that IJi6 marks the turning point of loop 2 from a continuous stacked conformation;
there are no NOES between U,, and U,,, and IJ16 adopts a C-2'-endo conformation.
The 3' side of stem 2 (Gzz through G,,) also forms a continuous A-like helix. There is more heterogeneity in the sugar conformation at the 3' end of stem 2 than in the nucleotides to which they are base-paired.
Both A,, and Gz6 have significant populations of S-type sugars, while Us and Cs have pure N-type sugar conformation.
On the 5' side of stem 1 there is some ambiguity in connectivities from Cz to G,; however, Cz and G3 are stacked in an A-like conformation.
The non-exchangeable and imino NOE data discussed above suggested that there was no significant perturbation in stacking between C,, and C,, at the junction of stem 1 and stem 2: where the two stem regions are covalently linked. However, the two helical regions do not appear to form a completely continuous A-like helix. There is no NOE connectivity between Gs and Gzz, G,, adopts primarily an S-type sugar pucker,, Even though they are not covalently linked, these nucleotides should exhibit the NOES characteristic of A-form stacking if the two stems were perfectly continuous.
The perturbation in helical structure must be minor. There is a NOE between imino protons on G, and Gzz (resonances 3 and 4, respectively) and the stacking between Cl2 and C,, appears A-like. As discussed below, this perturbation is not surprising since both C3 and Gz2 are involved in loop-helix junctions.
Information concerning the loop conformations in the pseudoknot is scant. Loop 1 is better defined than loop 2. Loop 1 crosses the deep major groove of stem 2. The 5' nucleotide in loop 1 (A4) shows no NOES to G,. The A, nucleotide may have a nonstandard conformation and is stacked upon U,. The far downfield chemical shift of A,(H8) may be indicat$ive of its environment within the loop. There are no NOES from U, to the unassigned U6. C, has a weak NOE to Hl', which probably belongs to U,. There may be stacking of U7 on Cs at the 5' terminus of stem 2, as suggested both by n.m.r. data and the double-strand-specific RNase V1 cleavage at this nucleotide. The residues of loop 1 are cleaved much more weakly by nuclease S, than loop 2 nucleotides, indicating some protection relative to single strands. Model building suggests that both A, and U,, even when stacked, can fit within the major groove. There is a possibility for tertiary interactions between the A, and the bases in stem 2 (i.e. U, or C,,). However, the N-7 atom of A, is more accessible to modification by DEP than either of the base-paired adenine residues. Thus, N-7 must be relatively accessible to solvent and not involved in hydrogen bonding. This does not, however, preclude interactions with other portions of the base ring. Alternatively, the loop region may be dynamically more flexible than the stem; the A,(HS) resonance is slightly broader than other resolved aromatic resonances.
Connecting loop 2 is poorly defined by the n.m.r. data. It consists of six uridine residues (U, 6-Uz 1), of which only U,, was definitely assigned. IJ16 is stacked at the 3' end of stem 1 (on Cis); there is no NOE connectivity to U1,. Ui6, as well as many of the uridine residues in the loop, adopts a south-type sugar conformation.
This loop crosses the minor groove of stem 1. Nuclease S, cleaves strongly after three loop residues, U1,, U1s and Uis. Loop 2 joins stem 2 at Gzz. The conformation of Gz2 is perturbed by the loop; it adopts a south-type sugar pucker. The S-type sugar pucker of U,, is adopted by nucleotides in tRNA at similar positions where the phosphodiester backbone changes direction. The n-turn observed in the anticodon loop of tRNAPh' results from changes in the values of the phosphodiester angles a and [ (Saenger, 1984) . This type of conformation may occur in the pseudoknot, but the values of the phosphodiester angles are probably gauche-gauche, rather than gauche-trans, since no peaks are shifted downfield in the "P n.m.r. spectrum (data not shown) (Gorenstein, 1981) .
(c) Structural model A low-resolution model of the structure of the pseudoknot has been constructed based on the results described above using the INSIGHT program on an Evans and Sutherland computer graphics terminal. The co-ordinates used for generation of the stems are based on an A-form helical structure from the fiber diffraction data of Arnott et al. (1973) . Loop regions were constructed to be consistent with the n.m.r. data. A detailed representation is shown in Figure 8 . The phosphate backbone from Cs to IJ16 is a continuous unperturbed A-form helix. On the opposite backbone, the two stem regions (G, to G, and Gzz to Gz6) are stacked in A-form geometry without distortion.
The region around Gs and Gzz contains close steric and electrostatic contacts between nucleotides from loop 2 and loop 1. It appears impossible to maintain a continuous, unperturbed helix without generating unfavorable interactions between phosphate molecules (highlighted in Fig. 9 ). The perturbation between G22 and G3 is therefore unsurprising.
Gzz adopts an S-type sugar conformation that, possibly coupled with other structural changes, may move the phosphates from loop 1 and 2 nucleotides further apart. A slight increase in the twist of the helix at the junction of Gzz and G3 would relieve further some of the close interactions between the two loops, while maintaining the coaxial stacking of the 'two helices. The electrostatic interactions in this region may also explain the requirement for divalent Figure 9 . View of the loop side of the pseudoknot model. Loop 1 crosses the major groove of stem 2 (lower) and loop 2 crosses the minor groove of stem 1 (upper). A, and U, in loop 1 are stacked within the major groove. Loop 2 is poorly defined by the n.m.r. data. In this undistorted model, the 2 loop regions can come into close contact. Two phosphate molecules with van der Waals' radii that come into particularly close contact are highlighted in red.
Loop 2 crosses the wide, shallow minor groove of stem 1. The distance (18 A) across this groove is longer than the distance across the major groove, even though only three base-pairs are spanned. Pseudoknot formation described here and variants with shorter loops (see the following paper) demonstrate that a loop size of six nucleotides is more than sufficient to bridge three base-pairs.
Most of the loop residues probably have an S-type sugar pucker. A south-type sugar enables a nucleotide to span a greater distance (7.0 A) than a nucleotide in a north conformation (5.9 A). The phosphodiester backbone probably changes direction between nucleotides U16 and U,,. There does not appear to be significant stacking in loop 2.
These results show unequivocally that RNA can form a pseudoknot structure with coaxial stacking of stems. The sequence studied here forms a single unimolecular structure in 5 mM:Mg'+; the-n.m.r. data are. not consistent with an equilibrium between structures. This possibility could not be excluded in our previous studies on pseudoknot sequences (Puglisi et al., 1988) . Since the pseudoknot structure forms in the absence of further stabilization from interactions with RNA outside of the pseudoknot region, pseudoknots are viable forms of local RNA structure.
The structural model consistent with the n.m.r. data is in good agreement with the original proposals of Pleij et al. (1985) and later model building studies on the TYMV RNA (Dumas et al., 1987) . The two stem regions stack in a coaxial geometry. Although data indicate some distortion from A-form stacking between stem 1 and stem 2 at the junction between stem and loop regions, the model is still consistent with the observed biological activity of the pseudoknots in the viral tRNA-like structures. The stacked stem regions of the pseudoknot structure in TYMV RNA mimics the seven base-pairs in the acceptor stem of tRNA"", allowing aminoacylation by valyl-tRNA synthetase (Pleij et al., 1985) . Florentz & Gieg& (1986) have mapped the interactions of valyl-tRNA synthetase with the TYMV RNA using chemical and enzymatic probes. The interactions with the "acceptor stem" formed by the pseudoknot are along the region equivalent to C, to C,,. It is this covalently connected phosphodiester backbone that will appear to the synthetase as a continuous A-form helix (Fig. 8) . Mutations in the base-pairs of a similar pseudoknot in brome mosaic virus RNA drastically reduced the aminoacylation by synthetase (Dreher & Hall, 19886) . Thus, a pseudoknot has two sides: one is a fairly undistorted A-form helix (the left side of the model shown in Fig. 8 ) and the other contains both loop regions as well as the junctions of the loops with the stems (Fig. 9) . Even if undistorted, the loop-junction side of the structure would not appear to a protein as an A-form helix.
The loop regions of the pseudoknot described in this study were purposely made long to allow them to span the stem regions easily; the effects of loop size on pseudoknot stability are described in the following paper. Structurally, the loop regions are less well characterized than the stems. Loop 1 has a better-defined structure than loop 2. The proposed distortion in the pseudoknot A-form geometry occurs in the region of the junction of the two loops with the stems (Fig. 9) . Distortion of the structure, mainly localized to the stack between G3 and GZ2 probably reduces unfavorable electrostatic interactions between phosphate groups at the loop-stem junctions.
Feat,ures of the pseudoknot other than the duplex region may be useful in biological functions. Proteins may recognize unique loop conformations. McPheeters et al. (1988) propose that bacteriophage T4 gene 32 protein regulates translation of its own mRNA by co-operative binding to the message. The nucleation site for the binding is proposed to be a pseudoknot.
The authors suggest that gene 3.2 protein recognizes a conformation adopted by the larger loop in their pseudoknot (equivalent, to loop 2). Since loop 2 is sterically less hindered than loop 1, it may be more accessible for protein binding. The structure studied here represents a specific> type of pseudoknot interaction; the stem regions are contiguous and stacked, and the loop regions arch relatively short. In general, pseudoknot interactions can form where the stems are not adjacent, An examination of the present structure shows that it one or two nucleotides are inserted between t,he two .
.
stem regions (1.e. between Cl2 and C1 3) close elect'rostatic interactions between the loops could be minimized although the coaxial stacking of the tw stems would be eliminated. In the proposed pseudoknot required for effcient frameshifting in coronavirus RNA the two stem regions are separated by a nucleotide (Brierley et al., 1989) . The loop regions of the pseudoknot can be made shorter as detailed in the following paper. or can be extremely long with secondary structurr of t'heil.
own. Pseudoknots with large loop sizes have been proposed in the structure of RNase P (James rt 01.. 1988). In 16 S rRNA, a pseudoknot is formed 1~~ pairing nucleotides 17 through 19 and 916 through 918 (Moazed & Noller, 1987) . Loop 2 of this pseudoknot contains most of the 16 S rRNA srcondar)structure. These long-range pseudoknots arc an effective mechanism for bringing regions distant, in sequence into close spatial proximity, Such a pseudoknot interaction has been proposed in the structural model for mitochondrial group I int,ronF (Davies et al., 1982) and may be recognized I)! specific proteins (Akins & Lambowitz. 198i: Delahodde et al., 1989) . In its versatile structures and functions RNA seems more akin to proteins than DNA. The results presented here demonstIratr the usefulness of n.m.r. studies on model oligonucleotides to determine biologically relevant R,N.+j structures.