Structure, Vol. 12, 237–248, February, 2004, 2004 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/j.str.2004.01.014
The Structure of the Stemloop D Subdomain of Coxsackievirus B3 Cloverleaf RNA and Its Interaction with the Proteinase 3C Oliver Ohlenschla¨ger,1,4 Jens Wo¨hnert,1,4 Enrico Bucci,1,2 Simone Seitz,3 Sabine Ha¨fner,1 Ramadurai Ramachandran,1 Roland Zell,3 and Matthias Go¨rlach1,* 1 Institut fu¨r Molekulare Biotechnologie e.V. Bentenbergstr. 100813 D-07745 Jena Germany 2 Istituto di Biostrutture e Bioimmagini Via Mezzocannone 6-8 I-80134 Napoli Italy 3 Institut fu¨r Virologie und Antivirale Therapie Friedrich-Schiller-Universita¨t Winzerlaer Straße 10 D-07745 Jena Germany
Summary Stemloop D (SLD) of the 5ⴕ cloverleaf RNA is the cognate ligand of the coxsackievirus B3 (CVB3) 3C proteinase (3Cpro). Both are indispensable components of the viral replication initiation complex. SLD is a structurally autonomous subunit of the 5ⴕ cloverleaf. The SLD structure was solved by NMR spectroscopy to an rms deviation of 0.66 A˚ (all heavy atoms). SLD contains a novel triple pyrimidine mismatch motif with a central Watson-Crick type C:U pair. SLD is capped by an apical uCACGg tetraloop adopting a structure highly similar to stable cUNCGg tetraloops. Binding of CVB3 3Cpro induces changes in NMR spectra for nucleotides adjacent to the triple pyrimidine mismatch and of the tetraloop implying them as sites of specific SLD:3Cpro interaction. The binding of 3Cpro to SLD requires the integrity of those structural elements, strongly suggesting that 3Cpro recognizes a structural motif instead of a specific sequence. Introduction Coxsackieviruses cause several acute or chronic human diseases, belong to the enterovirus genus of the Picornaviridae, and are closely related to polio- and rhinoviruses. Their genome consists of a positive-sense singlestranded RNA of ⵑ7.5 kb encoding a polyprotein of roughly 2200 amino acids which is processed by the viral proteases 2Apro, 3Cpro, and 3CDpro, the latter being the precursor of 3Cpro and the RNA polymerase 3Dpol. The genomic RNAs of entero- and rhinoviruses share a highly structured and evolutionary conserved 5⬘-nontranslated region (NTR) harboring the internal ribosomal entry site (IRES) required for translation and a 5⬘-terminal cloverleaf-like RNA element required for negative- and *Correspondence:
[email protected] 4 These authors contributed equally to this work.
positive-strand replication (Ehrenfeld and Teterina, 2002; Paul, 2002). Several viral proteins assemble upon the 5⬘ cloverleaf to form a ribonucleoprotein complex required for the initiation of the latter processes. In poliovirus, this 5⬘ ribonucleoprotein complex includes 3CDpro and the cellular poly(C) binding protein 2 (PCBP2) (Andino et al., 1990a, 1993; Gamarnik and Andino, 1997; Parsley et al., 1997) or the viral 3AB protein (Harris et al., 1994; Xiang et al., 1995). Cloverleaf-bound 3CDpro interaction with the cellular poly(A) binding protein bound to the 3⬘ poly(A) tract of the viral genomic RNA is important for the initiation of negative-strand synthesis (Barton et al., 2001; Herold and Andino, 2001; Teterina et al., 2001). The RNA binding activity of the 3CDpro was mapped to the 3C domain (Andino et al., 1990b), and 3Cpro alone recognizes specifically the 5⬘ cloverleaf in vitro (Leong et al., 1993; Zell et al., 2002). These data indicate that 3Cpro or the 3Cpro component of 3CDpro plays a crucial role in the assembly of the replication initiation complexes. The 5⬘ cloverleaf is highly conserved among all enteroand rhinoviruses. It consists of ⵑ90 nucleotides and contains four joint subdomains: stem A and the stemloops B, C, and D (Figure 1A). Subdomain D contains an asymmetric bulge at its base, a symmetric bulge bound by two base-paired stem regions and an apical loop, the D loop (Figures 1A and 1B), and is the cognate RNA ligand in vitro for 3Cpro or 3CDpro, respectively (Andino et al., 1990a; Walker et al., 1995; Zell et al., 2002). This notion is supported by in vivo data on a viable 3Cpro mutant suppressing a mutation in the D loop of poliovirus (Andino et al., 1990b). The viability of chimeric virus constructs with hybrid 5⬘ NTRs suggested that the 3Cpro/3CDpro:cloverleaf interaction is not serotype specific (Johnson and Semler, 1988; Todd et al., 1997; Xiang et al., 1995; Zell et al., 1995, 1999). Moreover, our in vitro analysis indicated that the presence of a 4 nucleotide D loop carrying the rather degenerate consensus sequence 5⬘-NNYR-3⬘ and flanked by a Y:G closing base pair is necessary and sufficient for specific binding of 3Cpro to stemloop D. This led to the proposal that structural features rather than the sequence of the apical D loop are a major specificity determinant for the 3Cpro:RNA interaction (Zell et al., 2002). Here we show that a 30-mer RNA (SLD) representing stemloop D of CVB3 containing nucleotides 50–76 of the 5⬘ cloverleaf (Figures 1B and 1C) is a structurally autonomous subdomain of the CVB3 5⬘ cloverleaf. The SLD structure contains a novel base-paired triple pyrimidine mismatch motif and indicates that the sequence space of tetraloops adopting the conformation of the stable cUNCGg is larger than expected. NMR analysis of the binding of 3Cpro to SLD shows that the D loop including its closing U:G base pair and two nucleotides adjacent to the central triple pyrimidine mismatch are interacting with 3Cpro. Supported by analysis of the RNAprotein interaction in the yeast three-hybrid system, our data strongly suggest that the RNA-protein recognition in this system is structure rather than sequence specific.
Structure 238
Results
The imino region of the 1H-15N correlation spectrum of SLD at 25⬚C was compared with the same region of a spectrum of the 15N-labeled cloverleaf RNA (Figure 2A). Signals corresponding to the imino groups of U14, G17, and G18 in the apical loop of SLD and to U6, U8, U23, and U25 in the central triple pyrimidine mismatch are detected for the complete cloverleaf at virtually the same chemical shifts as in the isolated SLD. The number of signals for G and U imino groups observed for the entire 5⬘ cloverleaf is consistent with its secondary structure (Figure 1B). In 1H-15N 2D correlation spectra of SLD at 10⬚C (Figure 2B), all imino resonances, including for U24, were detected. The signal of the imino group of G17 located in the D loop appears at 10.73 ppm, indicating its involvement in a nonstandard hydrogen bond. The presence of imino resonances for all five U residues (U6, U8, and U23–U25) of the central triple pyrimidine mismatch of SLD implies a well-defined local structure. Except for U24, all these resonances are detected up to 35⬚C, but the imino resonances of U13, G17, and G18 in the apical loop and U8 and U25 of the central triple pyrimidine mismatch become severely broadened at 45⬚C (data not shown). Hydrogen bonding in SLD as analyzed by HNN-COSY experiments (Dingley and Grzesiek, 1998) revealed eight correlations between U or G imino groups and the N1 of A and the N3 of C, respectively (Figure 2B), consistent with eight canonical A:U and G:C pairs in the secondary structure (Figure 1C). A correlation between the imino proton of U24 and the N3 of C7 (Figure 2B) indicated an unexpected direct hydrogen bond of a Watson-Crick type C:U base pair at the center of the central triple pyrimidine mismatch. The 2hJN3N3 coupling constant of 5.3 Hz is comparable to values observed elsewhere (Dingley and Grzesiek, 1998; Wo¨hnert et al., 1999). The 13C chemical shifts of uridine carbonyl C2 and C4 nuclei of U6, U8, U23, and U25 were compared to the carbonyl chemical shift of uridines in A:U and in wobble G:U base pairs observed in the H(N)CO experiment. The systematic deviation of those chemical shifts indicated hydrogen bonds typical for two asymmetric U:U base pairs (see also Theimer et al., 2003).
Structure Determination Recently, we demonstrated (Zell et al., 2002) that 3Cpro forms a stable complex with the entire CVB3 5⬘ cloverleaf (Figure 1B) and stemloop D (SLD; Figure 1C) comprising nucleotides 50–76 of the cloverleaf. The affinity of 3Cpro for SLD (KD ⵑ2 M) and for the entire cloverleaf (KD ⵑ1 M) are very similar (data not shown) (Zell et al., 2002). For structure determination, SLD (Figure 1C) was produced by in vitro transcription in uniformly 15N-, 15 N/13C-, and in G,C- or A,U-base specifically [15N,13C]labeled form. An entire 15N-labeled 5⬘ cloverleaf was also prepared. Virtually complete resonance assignments for SLD were achieved by standard triple resonance experiments augmented by new experiments for the assignment of pyrimidine base spin systems (Wo¨hnert et al., 2003). The G,C- and A,U-[15N,13C]-labeled samples allowed for unambiguous assignments of ribose moieties and for the identification of important intra- and internucleotide NOEs.
Overall Structure of the SLD The SLD structure was calculated on the basis of 1001 NOE constraints derived from 15N- and 13C-edited NOESY spectra using 15N- and G,C-, or A,U-[15N,13C]-labeled samples. The ensemble of the energy minimized structures with the lowest target function is shown in Figure 3A. The structural statistics is given in Table 1. SLD shows a compact structure with nucleotides G1–U13 and G18–C30 adopting an all-helical conformation. Two regular A-helical elements bound a central base-paired triple pyrimidine (U6:U25-C7:U24-U8:U23) mismatch (Figure 3B). The helical stem is capped by a well-defined tetraloop structure closed by the U13:G18 wobble base pair (Figure 3C). All nucleotides adopt C3⬘-endo ribose conformation except for A15 and C16 which are in C2⬘endo conformation. A strong intraresidue H1⬘–H8 NOE cross peak indicated a syn glycosidic conformation for G17.
Figure 1. The 5⬘ NTR of CVB3 (A) Schematic representation of the entire 5⬘ NTR of the CVB3 genomic RNA; stemloop D (SLD) is highlighted by a gray box. (B) The 5⬘ cloverleaf of CVB3 (nt 1–104); the boxed region corresponds to SLD. (C) Secondary structure of SLD. Nucleotides 3–28 (bold) correspond to positions 51–76 of the 5⬘ cloverleaf; terminal nucleotides (italics) were added for in vitro transcription.
Stemloop D-Proteinase 3C Interaction 239
Figure 2. 1H-15N Correlation Spectra of SLD and the 5⬘ Cloverleaf of CVB3 (A) Superimposed 1H-15N HSQC spectra of SLD (closed contours) and the 15N-labeled entire 5⬘ cloverleaf (open contours) at 298 K. Assignments for imino protons of SLD are indicated. Positions marked with an asterisk correspond to the terminal nucleotides of SLD. (B) HNN-COSY spectrum of 15N-labeled SLD at 283 K. Assignments and the typical 15N chemical shift regions for the N1 of G, N3 of U, N3 of C, and N1 of A are indicated. Dotted lines connect cross peaks arising from NH···N hydrogen bonds of Watson-Crick base pairs. The solid line connects the cross peaks arising from the NH···N hydrogen bond between the imino group of U24 and the N3 nitrogen of C7.
Structure of the Triple Pyrimidine Mismatch The central triple pyrimidine mismatch of the SLD stem (Figure 3B) consists of two asymmetric U:U base pairs flanking a central C:U pair (Figure 4A). Hydrogen bonds are formed between the imino function of U6/U23 and the C4 carbonyl group of U25/U8 and between the imino group of U25/U8 and the C2 carbonyls of U6/U23, respectively (Figure 4A). The central Watson-Crick-type C7:U24 pair forms two hydrogen bonds with distances of about 2 A˚. The distance between the N3 of C7 and the imino group of U24 accommodates a direct hydrogen bond and is virtually identical to the corresponding distances in the canonical A:U or G:C base pairs of SLD. The triple pyrimidine mismatch exhibits a narrowing of the minor groove with C1⬘-C1⬘ distances reduced by ⵑ2 A˚ compared to regular A helix geometry (Figure 4A). No intrastrand base-base stacking is seen for the mismatched pyrimidines. Only stacking of the ring substituents is observed (Figure 4C). The C5:G26-U6:U25 step is a mirror image of the U8:U23-G9:C22 step (Figures 4B and 4D). The base moieties of C5 and C22 stack on top of the U6 and U23 base while the stacking of U8 and U25 onto G9 and G26, respectively, is less pronounced (Figures 4B and 4D). The triple pyrimidine mismatch projects six carbonyl functions into the minor groove and five carbonyl functions into the major groove. This creates patches of considerable electronegativity but also of potential hydrogen bond acceptors in this part of the structure.
Structure of the Apical D Loop The apical uCACGg D loop has a well-defined structure (Figure 3C) and is closed by the U13:G18 wobble base pair inducing a widening of the major groove at the loop base. The first and last nucleotides of the loop (C14 and synG17) form an unusual base pair (Figure 5A). The structure indicates the formation of hydrogen bonds involving the O6 of G17 and the 2⬘OH of C14 as well as the imino proton and the amino group of G17 and the O2 of C14 (Figure 5A). In agreement with the hydrogenbonding pattern, a resonance assigned to the 2⬘OH proton of C14 was observed at 10⬚C at 6.66 ppm, yielding NOE correlations to its own H1⬘ and H2⬘ as well as to the H2⬘ of A15, H1⬘ of G17 and H3⬘ of U13. The second and third loop nucleotides (A15 and C16) adopt C2⬘endo ribose conformation. The A15 base moiety protrudes into the minor groove and C16, C14, and U13 form a continuous stacking motif on the major groove side (Figure 5B). C14 establishes a base to backbone hydrogen bond from its amino function to a nonbridging oxygen of the phosphate group of A15. Albeit sequence and closing base pair of the D loop differ from known stable tetraloops (Allain and Varani, 1995b; Ennifar et al., 2000), its structure is virtually indistinguishable from that of the canonical cUNCGg tetraloop. The backbone of the D loop can be superimposed upon the backbone of the stable cUNCGg tetraloops (Allain and Varani, 1995b; Ennifar et al., 2000) with an rmsd of 0.80 and 0.69 A˚, respectively (Figures 5C and 5D). The structural
Structure 240
Figure 3. NMR Solution Structure of the SLD from CVB3 (A) Stereo view of a superimposition of 40 energy-minimized of 100 calculated structures. A-helical residues are in black; and residues of the noncanonical parts of the structure are color coded: U (yellow), C (blue), G (green), and A (brown). (B) Local superimposition of the U6:U25-C7:U24-U8:U23 noncanonical base pairs and their flanking G:C pairs; view into the major groove; color coding as in (A). (C) Local superimposition of the apical tetraloop structure and the closing U13:G18 base pair; the major groove aspect is shown; color coding is as in (A).
homology between the D loop and the cUUCGg stable tetraloop is reflected by the similar signature of unusual chemical shifts (Allain and Varani, 1995a) for the H1⬘/ C1⬘ of A15, C16, G17, and G18; the H2⬘/C2⬘ of C14, A15, C16, and G17; the H3⬘/C3⬘ of A15, C16, and G17; for H4⬘/C4⬘ of A15 and C16; and for the H5⬘/H5″ of C16, G17, and G18, as well as for the H8/C8 of the synG17, respectively.
Binding of 3Cpro to SLD To analyze the interaction of SLD with 3Cpro, 1H-15N HSQC spectra for the imino region and 1H-13C HSQC spectra for aromatic resonances were recorded for the free SLD and for a SLD:3Cpro complex. Due to limited solubility of 3Cpro, the spectra had to be taken on dilute samples containing only 80 M RNA and 300 mM KCl. Comparison of the 1H-15N HSQC spectra of the free SLD and of
Stemloop D-Proteinase 3C Interaction 241
Table 1. Structural Statistics for the Stemloop D RNA Experimental constraints Distance constraints 1001 Torsion angle constraints 258 a Hydrogen bond constraints 52 CYANA target function before 0.25 ⫾ 0.03 energy minimization (A˚2) AMBER energies (kcal/mol) after energy minimization Physical energy ⫺184.20 ⫾ 9.25 Van der Waals energy ⫺343.70 ⫾ 3.33 Electrostatic energy ⫺560.91 ⫾ 6.12 Number of violations NOE viol. ⱖ0.2 A˚ 0 Torsion angle restraint viol. ⱖ2.5⬚ 0 Mean deviation from ideal covalent geometry Bond length (A˚) 0.0047 ⫾ 0.000085 Bond angles (⬚) 1.48 ⫾ 0.02 Heavy atom rms deviation from mean structure (A˚) All 0.66 Tetraloop (U13–G18) 0.27 Mismatch (U6–U8; U23–U25) 0.16 Best to mean 0.31 a
36 in initial rounds and 52 in final rounds of structure calculation; see Experimental Procedures
the SLD:3Cpro complex with an RNA:protein ratio of 1:1.1 reveals a shifted imino resonance for U13 and broadening of the G17 and G18 imino resonances beyond detection (Figure 6A). Addition of 3Cpro up to 1.3-fold excess over SLD did not result in further spectral changes. In the 1H-13C HSQC spectrum for the aromatic region of the 3Cpro-bound SLD, the signals of H8/C8 of G17; H8/ C8 and H2/C2 of A15; and H6/C6 of C5, U13, C14, C16, and C21 are extremely broadened or absent (Figure 6B). No change of chemical shifts or line broadening was observed for the respective resonances of other nucleotides. Six affected residues (U13–G18) belong to the D loop and its closing wobble base pair. Two residues (C5 and C21) reside adjacent to the central triple pyrimidine mismatch facing the same side of the helical stem region of SLD (Figure 8). Interaction of Stemloop D Variants and 3Cpro The 3Cpro binding to RNA was further characterized using the yeast three-hybrid system (SenGupta et al., 1996). The 5⬘ cloverleaf and SLD bind 3Cpro (Figure 7A). For a 3CDpro fusion construct, binding was detected as for 3Cpro alone. Deletion of C64 (corresponding to C16 in
Figure 4. Structural Details of the Central Triple Pyrimidine Mismatch of SLD (A) Local superimposition of the base pairs of the central triple pyrimidine mismatch and its flanking canonical base pairs. Hydrogen bonds are indicated by dotted lines; the hydrogen bond between the N3 nitrogen of C7 and the imino proton of U24 is highlighted in yellow. The C1⬘-C1⬘ distances are indicated. (B–D) Stacking pattern for the central mismatch and its flanking base pairs. (B) Stacking of the G9:C22 flanking base pair onto the asymmetric U8:U23 pair. (C) Stacking of the three noncanonical base pairs of the central triple pyrimidine mismatch. (D) Stacking of the asymmetric U6:U25 pair onto the flanking C5:G26 base pair.
Structure 242
Figure 5. Structural Details of the Apical Tetraloop of SLD (A) The noncanonical C14:synG17 base pair. (B) Triple pyrimidine stack of the apical tetraloop and the closing U13:G18 base pair. Hydrogen atoms and base atoms of A15 (yellow) are omitted for clarity. (C and D) Superimpositions (side view and top view) of the apical tetraloop of SLD (red), of the canonical tetraloop cUUCGg from the P1 element of the group I self-splicing intron (blue, PDB 1HLX), the TL1 cUUCGg tetraloop from 16S rRNA (yellow, PDB 1F7Y), and the apical triloop of the stemloop D of HRV14 (gray, PDB 1IK1). For the superimposition of the HRV14 triloop onto the tetraloops, the respective closing base pairs were used as reference.
SLD) from the D loop of the 5⬘ cloverleaf abolishes binding (Figure 7A). The wild-type HRV14 cloverleaf with a 3 nucleotide D loop does not bind the CVB3 3Cpro, but the insertion of a single G into the D loop enables binding. 3Cpro binding was also detected when the D loop in the CVB3 cloverleaf was replaced by the poliovirus sequence (uUGCGg) or by the same loop closed by a C:G base pair (cUGCGg), the latter representing a canonical stable tetraloop of the cUNCGg-type (Figure 7A). Analysis of homonuclear 1D and 2D NOESY spectra of a HRV14 SLD mutant containing a cUAUGg D loop, thereby corresponding to the HRV14InsG60 cloverleaf mutant, as well as the CVB3 SLD containing the PV1 D loop reveals imino resonances around 10 ppm (Figure 7B) also for those mutant RNAs. Such a chemical shift is indicative for the synG nucleotide in UNCG-type tetraloops (Allain and Varani, 1995b; Proctor et al., 2002) and corresponds to the imino signal of the synG17 of the CVB3 D loop. This strongly suggests a cUNCGgtype tetraloop conformation also for these 3Cpro binding mutants. In addition, the importance of the central U6:U25-C7:U24-U8:U23 mismatch for binding of 3Cpro was demonstrated by deletion of U23 which abolishes binding. However, replacing the central C7 by an U and thereby creating a triple U:U mismatch, not found in any viral sequence yet, did not impair binding (Figure 7A). A 3Cpro containing a mutated KFRDI motif important for RNA binding (Leong et al., 1993; Matthews et al., 1994;
Walker et al., 1995) did not bind (data not shown) as expected. Discussion Stemloop D (SLD) of CVB3 is highly homologous to all SLDs of entero- and rhinoviruses (Zell et al., 2002; Zell and Stelzner, 1997) and serves as the essential and cognate RNA ligand for 3Cpro in the viral replication process. Comparison of 1H-15N correlation spectra recorded on SLD and the 5⬘ cloverleaf (Figure 2A) shows virtually identical chemical shifts for imino resonances of nucleotides of the D loop and the internal symmetric mismatch. This indicates that the structure of SLD in isolation is very similar to the structure of stemloop D in the context of the whole cloverleaf. Hence, long-range tertiary interactions between the internal symmetric mismatch or the apical loop of SLD and the remainder of the cloverleaf appear to be absent. Thus, SLD represents a structurally autonomous subdomain of the viral cloverleaf. The basis of the SLD:3Cpro interaction was investigated by solving the structure of SLD and by characterizing its interaction with 3Cpro using NMR spectroscopy and the yeast three-hybrid system. The SLD contains two prominent structural motifs, a new base-paired U:UC:U-U:U mismatch motif situated at the center of the stem of SLD and a well-structured tetraloop resting upon a U:G wobble base pair.
Stemloop D-Proteinase 3C Interaction 243
and the O4 of U24 as well as between the N3 nitrogen of C7 and the imino proton of U24. In contrast, hitherto observed C:U base pairs involve one direct and one water-mediated hydrogen bond and exhibit an A-helical C1⬘-C1⬘ distance (Holbrook et al., 1991; Tanaka et al., 2000; Theimer et al., 2003; Lescrinier et al., 2003). In SLD, the C1⬘-C1⬘ distance for the two U:U base pairs and the C7:U24 base pair is reduced by about 2 A˚ compared to A-helical values (Figure 4A) which accommodates the smaller size of the bases as compared to a canonical Y:R pair. The only other example for such a C:U base pair in a different structural context mediating a long-range RNA-RNA interaction we found in the structure of the 50S ribosomal subunit from H. marismortui (C1545:U1702 [Ban et al., 2000]) using HBexplore (Lindauer et al., 1996). This C:U pair geometry causes a 3 A˚ distance of the C2 carbonyl oxygen atoms. However, the resulting electrostatic clash is possibly alleviated by incorporating water molecules or a metal cation as observed, e.g., in the group I intron (Basu et al., 1998; Cate et al., 1997) and the signal recognition particle RNA (Batey and Doudna, 2002). A coordination of cations by the mismatch region would also contribute to neutralizing the negative charge density produced by the six carbonyl oxygens in the minor groove and the five carbonyl groups in the major groove. The central triple pyrimidine mismatch is flanked by canonical C:G pairs. The base pair geometry of the asymmetric U:U pairs and their stacking pattern onto the flanking C:G pairs (Figures 4B and 4D) is virtually identical to that of a duplex RNA with a central C:G-U:U-U:U-G:C motif (Lietzke et al., 1996).
Figure 6. Binding of 3Cpro to SLD RNA NMR spectra of 15N/13C-labeled SLD were recorded in the absence (single heavy contour) or the presence of a 1.1-fold excess of 3Cpro (thin contours) at a concentration of 80 M RNA. Assignments for the free SLD are indicated. (A) Superimposition of the imino region of 1H-15N HSQC spectra. (B) Superimposition of the H6C6, H8C8, and H2C2 (inset) regions of 1H-13C HSQC spectra. The relevant assignments in the H6C6 region are indicated.
The Central Triple Pyrimidine Mismatch The U6:U25-C7:U24-U8:U23 mismatch region of SLD represents a novel structural feature for RNA. Although uridines are frequently involved in base-paired mismatches (e.g., Lietzke et al., 1996; Jiang et al., 1997, 1999; Heus and Hilbers, 2003), the only other example for a SLD-like pyrimidine-rich base-paired mismatch constitutes the recent structure of a telomerase RNA encompassing three consecutive asymmetric U:U pairs and a water-mediated U:C pair (Theimer et al., 2003). However, the mismatch of SLD contains two asymmetric U:U base pairs flanking a central C:U pair. All three base pairs form two direct hydrogen bonds (Figure 4A). The central C:U pair is of the Watson-Crick type with hydrogen bonds between the exocyclic amino group of C7
The D Loop Structure Despite its different sequence and closing base pair, the D loop structure is very similar in overall geometry and hydrogen bonding to the canonical cUNCGg tetraloop (Allain and Varani, 1995b; Ennifar et al., 2000). This includes the noncanonical base pair between the first and the fourth loop nucleotide in syn conformation (C14:synG17) including a hydrogen bond involving the 2⬘OH of C14, the continuous stack formed by the third and fourth base of the loop and the pyrimidine of the closing pair (Figure 5), and a hydrogen bond between the exocyclic amino group of the third loop base (C16) and a nonbridging oxygen of the C14-p-A15 phosphate. A partial structure of SLD including the D loop is very similar to the corresponding part of our structure as judged from the recently published figures and data (Du et al., 2003). Recently, the sequence space of thermostable cUNCGg-type tetraloops was extended to cYNMGg (Proctor et al., 2002). The SLD structure and the initial results on SLD mutants (Figure 7B) show that an even greater number of sequences is able to adopt a cUNCGglike conformation as the D loop is closed by an U:G wobble base pair, whereas the canonical YNMG loop is closed by a C:G pair. This provides structural proof for an extended family of such tetraloops. However, the severe broadening of the imino proton resonances of U13, G17, and G18 at 45⬚C indicates a lower melting temperature for the D loop as compared to the cYNMGg stable tetraloop family (Proctor et al., 2002). Given the
Structure 244
Figure 7. Binding of 3Cpro to Cloverleaf Mutants and NMR Characterization of SLD Mutants (A) Binding of 3Cpro and 3CDpro to wild-type and mutant CVB3 and HRV14 cloverleaf (CL) and SLD RNA, respectively, in the yeast three-hybrid system. The iron-responsive protein (IRP) and the iron-responsive element RNA (IRE) were used as control. WT, wildtype RNA fused to the MS2 phage carrier RNA. Deleted nucleotides are indicated by a ⌬ and their positions in the cloverleaf. C55U, C7 to U7 mutant of the central triple pyrimidine mismatch of the SLD of CVB3. cUGCGg, the D loop of CVB3 replaced by a stable tetraloop. UUGCGg, poliovirus 1 D loop in the context of the CVB3 cloverleaf. Ins G60, insertion of a G in position 60 of the HRV14 cloverleaf. (B) 1D 1H NMR imino spectra (detail) of RNAs representing CVB3 SLD (SLD), poliovirus 1 D loop in the context of the CVB3 SLD (uUGCGg), and the shortened HRV14 SLD InsG60 (Ins G60) mutant recorded at 15⬚C. Assignments derived from 1H-1H 2D NOE spectra are indicated. Imino resonances arising from duplex forms of the mutant RNAs (“D”), even though folding and recording of spectra was done at only 10 mM KCl to minimize duplex formation.
high similarity in structure and hydrogen bonding between the D loop and the stable cUNCGg tetraloop, this adds further direct evidence to the importance of the closing base pair for the thermostability of such tetraloops. Reverting the C:G closing base pair to a G:C pair reduces the Tm of thermostable tetraloops dramatically (Antao et al., 1991; Proctor et al., 2002). The U:G closing pair here induces a local widening of the major groove. The increase by 0.4 A˚ of the U13C1⬘–G18C1⬘ distance reduces slightly the overall stacking between the D loop C14:synG17 base pair and the closing U13:G18 base pair as compared to the respective step of the stable tetraloop (Allain and Varani, 1995b; Ennifar et al., 2000). In view of this, we propose that the U13:G18 closing wobble base pair is reducing the thermostability of the D loop. 3Cpro Binding to SLD The affinity of 3Cpro for SLD and for the entire cloverleaf in vitro is very similar (Zell et al., 2002), suggesting that other elements of the CVB3 5⬘ cloverleaf do not contribute significantly to the specificity of this RNA:protein recognition process, albeit additional interactions of the SLD:3Cpro complex with other viral or cellular proteins, e.g., the PCBP2 (Gamarnik and Andino, 1997; Parsley et al., 1997), during the assembly of the replication complex cannot be ruled out. 3Cpro binds to SLD as well as to the entire cloverleaf in the yeast three-hybrid system (Figure 7), indicating that SLD is a major recognition element for 3Cpro binding. This is consistent with results obtained from a mammalian cell-based system for the PV1 3Cpro (Blair et al., 1998) and with the observation of a viable polioviral 3Cpro second site revertant (Andino et al., 1990b). Nevertheless, dissecting the SLD:3Cpro interaction in more detail is crucial to gain insight into the
mechanism of the entero-rhinoviral replication complex assembly. Hence, we investigated the interaction of CVB3 3Cpro with SLD by NMR methods (Figure 6) complemented by a mutational analysis of cloverleaf variants in the yeast three-hybrid system (Figure 7). RNA-protein complexes can be characterized by comparing NMR spectra of the free binding partners with spectra of the respective complex (Go¨rlach et al., 1992; Stoldt et al., 1999; Lebars et al., 2003). The interaction of 3Cpro with SLD was analyzed using 1H-15N and 1 H-13C HSQC spectra of SLD in the absence and the presence in excess, respectively, of 3Cpro (Figure 6). Signals of the imino groups of U13, G17, and G18 and of several aromatic protons (see Results) were broadened beyond detection or shifted in the presence of 3Cpro. This line broadening is indicative of the 3Cpro:SLD complex being at an intermediate exchange on the NMR time scale consistent with the micromolar affinity of 3Cpro for SLD (Zell et al., 2002). The affected residues of SLD are shown in Figure 8. The results provide experimental evidence that interactions between SLD and 3Cpro occur with the D loop and its closing U13:G18 base pair. The local widening of the major groove induced by the U13:G18 wobble pair is less likely to be as significant for binding as in other systems (Cai et al., 1998; Legault et al., 1998) since for SLD this base pair can be replaced by the canonical C:G closing base pair without impairing the binding of 3Cpro (Figure 7A). As a U:G or a C:G closing base pair projects a similar pattern of hydrogen bond donors and acceptors into the minor groove, this face of the D loop could be contacted by 3Cpro together with the conserved synG17. Two additional residues adjacent to the central triple pyrimidine mismatch (C5 and C21) are implicated in 3Cpro binding (Figures 6 and 8) and are presenting their major groove edge at the same
Stemloop D-Proteinase 3C Interaction 245
contrast, a chimera carrying a D loop with three nucleotides and not able to adopt this fold (Huang et al., 2001) (Figures 5C and 5D) is not viable (Xiang et al., 1995). The results presented here explain the interaction of 3Cpro with apparently degenerate tetraloop sequences in structural terms. They also provide structural evidence that a larger number of RNA sequences may adopt a cUNCGg-like tetraloop conformation. Mutation and genomic recombination are common events in picornaviruses and the preferred site of recombination localizes to the 5⬘ NTR (Santti et al., 1999). The sequence conservation of SLDs is relatively high, yet the D loop is the least conserved element (Zell et al., 2002; Zell and Stelzner, 1997). In view of the present data, it is suggested that the SLD:3Cpro recognition is structure based in entero- and rhinoviruses. Such a mechanism would provide a “safety strategy” with an evolutionary advantage for the replication of the continuously changing viral genome. Experimental Procedures Figure 8. Nucleotides of SLD Involved in 3Cpro Binding CPK model and secondary structure of SLD are shown. Nucleotides of SLD interacting with 3Cpro as deduced from the NMR experiments are shown in light gray. The remainder of the nucleotides is in dark gray.
face of SLD. As the minor groove side of the D loop is located on this same face of SLD (Figure 8), it appears that this side of the D loop and the major groove at C5 and C21 form a topologically interdependent bipartite recognition site for 3Cpro. The NMR data do not reveal a direct interaction between the central pyrimidine mismatch and 3Cpro. However, the loss of binding caused by an U23 deletion in the yeast three-hybrid system (Figure 7A) clearly points toward an important role of the central pyrimidine mismatch in the recognition process. Interestingly, the replacement of the central C7:U24 base pair by an U:U pair, most likely preserving the narrowed helical conformation (Theimer et al., 2003) in the central mismatch, preserves binding (Figure 7A). Hence, the particular shape of the helix at and adjacent to the central triple pyrimidine mismatch appears important for 3Cpro binding. Furthermore, the NMR data (Figure 6A) indicate that the base pairing in the helix of SLD including the central pyrimidine mismatch is not disrupted and that the U:G wobble closing base pair of the D loop is preserved in the complex. Since 3Cpro binds to a cloverleaf variant with a thermostable cUNCGg loop in the yeast threehybrid system (Figure 7A) and in vitro (Zell et al., 2002), it appears energetically unfavorable to significantly change the loop conformation as a prerequisite for binding 3Cpro. This is consistent with the notion that essentially all sequences binding CVB3 3Cpro in vitro conform to the sequence requirements necessary to adopt the conformation of cUNCGg tetraloops (Figure 7B) (Zell et al., 2002). Moreover, chimeric viral RNA constructs support viral growth in vivo as long as the SLD contained conforms to this sequence requirement (Johnson and Semler, 1988; Xiang et al., 1995; Zell et al., 1995). In
Sample Preparation Stemloop D RNA (SLD) from CVB3 was prepared and purified in unlabeled, uniformly 15N-labeled form, and uniformly 15N/13C-, G,C[15N,13C]-, and A,U-[15N,13C]-labeled form in vitro as described (Stoldt et al., 1999; Zell et al., 2002). The 104-mer representing the entire cloverleaf was prepared in uniformly 15N-labeled form. Final concentrations were 0.9 mM U-[15N,13C]-SLD, 1.2 mM G,C-[15N,13C]-SLD, 1.2 mM A,U-[15N,13C]-SLD, 1.4 mM U-[15N]-SLD, and 0.5 mM U-[15N] cloverleaf in 10 mM KH2PO4/K2HPO4, pH 6.2, with 40 mM KCl, 0.2 mM EDTA, and 5% v/v 2H2O or 99.99% v/v 2H2O, respectively. To compare the NMR spectra of the RNA in its free form and bound to 3Cpro, the U-[15N,13C]-SLD-RNA was exchanged into 20 mM KOAc, pH 5.5, 300 mM KCl buffer, and a 80 M sample of the free RNA and of the RNA-protein complex were prepared. A 20-mer HRV14 SLD mutant containing a cUAUGg D loop and the CVB3 SLD mutant containing the PV1 D loop were chemically synthesized (MWG Biotech, Munich, Germany), deprotected, and purified according to the manufacturer’s protocol. Overexpression and purification of proteolytically active 3Cpro was performed as described (Zell et al., 2002). The protein was exchanged into NMR buffer (20 mM KOAc, pH 5.5, 300 mM KCl) by dialysis and concentrated to a final concentration of 110 M. NMR Spectroscopy and Spectral Assignments NMR spectra were acquired on Varian UNITYINOVA 600 MHz or 750 MHz spectrometers. Data were processed with VNMR (Varian Inc., Palo Alto, CA) and analyzed with XEASY (Bartels et al., 1995). Measurements were performed at 298 K and at 283 K for the assignment and the detection of NOEs of exchangeable protons. Ribose spin systems were identified by a combination of 3DHCCH-COSY, -RELAY-COSY, and -TOCSY experiments (Nikonowicz and Pardi, 1993) recorded on the U-[15N,13C]-SLD sample and a HCCH-TOCSY experiment on the G,C-[15N,13C]-SLD sample. Base spin systems of pyrimidines were identified using H6/H5(C4N)H and C6/C5(C4N)H experiments (Wo¨hnert et al., 2003), guanine base spin systems using a modified H(CCN)H-TOCSY experiment (Sklenar et al., 1996), and adenine base spin systems using a HCCH-TOCSY experiment (Marino et al., 1996; Nikonowicz et al., 1992). C2 and C4 resonances were assigned by a 2D H(N)CO experiment. Sequential assignments and the linkage between base and ribose moieties were obtained as described (Stoldt et al., 1999). Guanine and adenine amino resonances were assigned using sequential and intra base pair amino/imino NOE connectivities in 2D 1H-15N CPMGHSQC-NOESY (Mueller et al., 1995) experiment. Resonance assignments are essentially complete with the exception of the amino group of A15 and for the H5⬘/C5⬘ or H5″/C5⬘ of A4 and of U11, 13, 19, 24, and 29.
Structure 246
Distance and Dihedral Angle Constraints for Structure Calculations NOE constraints were taken from 3D 1H-1H-13C NOESY-HSQC and 3D 13C-F1-filtered, 13C-F3-edited NOESY-HSQC spectra (Zwahlen et al., 1997) recorded in D2O (150 ms mixing time) using a G,C-[13C,15N]SLD and a A,U-[15N,13C]-SLD at 298 K. NOEs involving imino/amino resonances were derived from 2D 1H-1H NOESY, 2D 1H-15N CPMGNOESY (Mueller et al., 1995), and 3D 1H-1H-15N NOESY-HSQC (100 ms mixing time) spectra recorded at 283 K in H2O with the U-[15N]SLD sample. Upper limit distance constraints for the nonexchangeable hydrogens were classified according to their intensity in the NOESY spectra corresponding to distance limits of 2.8, 3.8, and 5.8 A˚, respectively. For the exchangeable hydrogens, only two classes corresponding to 3.3 and 5.8 A˚ were introduced. A lower distance limit of 1.8 A˚ was used for all classes. NOE intensities corresponding to fixed H5-H6 distances and intra base pair HN to H2 distances were used for calibration. Backbone torsion angles ␣ and of residues 1–12 and 19–30 were restricted to a range of ⫾60⬚ of A-helical values since no downfieldshifted 31P resonances were detected in the 31P NMR spectrum (Gorenstein, 1984; Varani et al., 1991). Due to the absence of H1⬘/ H2⬘-cross peaks in a CLEAN-TOCSY experiment (Griesinger et al., 1988; Kolk et al., 1998) with 20 ms mixing time and a spin-lock field of 10 kHz, the sugar pucker was restricted to C3⬘-endo for nucleotides 1–14 and 17–30 and to C2⬘-endo for A16 and C17 as strong H1⬘/H2⬘-cross peaks were observed for these nucleotides in the same experiment. Constraints for the torsion angles  and ⑀ for guanine or cytosine nucleotides were derived from 2D {31P}-spinecho-difference-CT-HMQC- and HSQC experiments (Hu et al., 1999; Legault et al., 1995; Szyperski et al., 1999) recorded with a constant time of 24 ms on the G,C-[15N,13C]-labeled sample in D2O. Further torsion angle constraints, e.g., for the glycosidic torsion angle of residues 1–8 and 10–30, were obtained from a local conformational analysis with the FOUND module (Gu¨ntert et al., 1998) in CYANA.
Acknowledgments The authors thank J. Su¨hnel for the help with HBexplore, T. Munder for helpful discussions, M. Stoldt, B. Heise, and H. Schwalbe for critically reading the manuscript, and A. Heller and S. Wachsmuth for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Go 474/3-1, DFG Ze 446/1-1). Received: June 4, 2003 Revised: October 24, 2003 Accepted: October 24, 2003 Published: February 10, 2004 References Allain, F.H., and Varani, G. (1995a). Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns. Nucleic Acids Res. 23, 341–350. Allain, F.H., and Varani, G. (1995b). Structure of the P1 helix from group I self-splicing introns. J. Mol. Biol. 250, 333–353. Andino, R., Rieckhof, G.E., and Baltimore, D. (1990a). A functional ribonucleoprotein complex forms around the 5⬘ end of poliovirus RNA. Cell 63, 369–380. Andino, R., Rieckhof, G.E., Trono, D., and Baltimore, D. (1990b). Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5⬘ noncoding region. J. Virol. 64, 607–612. Andino, R., Rieckhof, G.E., Achacoso, P.L., and Baltimore, D. (1993). Poliovirus RNA synthesis utilizes an RNP complex formed around the 5⬘-end of viral RNA. EMBO J. 12, 3587–3598. Antao, V.P., Lai, S.Y., and Tinoco, I., Jr. (1991). A thermodynamic study of unusually stable RNA and DNA hairpins. Nucleic Acids Res. 19, 5901–5905. Ban, N., Nissen, P., Hansen, J., Moore, P.B., and Steitz, T.A. (2000). The complete atomic structure of the large ribosomal subunit at 2.4 A˚ resolution. Science 289, 905–920.
Structure Calculation and Coordinates 1001 experimental distances derived from NOE cross peaks were used as upper limit constraints in CYANA (Herrmann et al., 2002). In initial structure calculations a total of 36 hydrogen bond constraints (four upper and four lower limit constraints/base pair) were used for base pairs for which an HN···N hydrogen bond was detected directly by the HNN-COSY experiment (Dingley and Grzesiek, 1998). Additional hydrogen bond constraints reflecting the hydrogen bonds forming consistently during the initial structure calculations for the G:U wobble base pairs (2:29 and 13:18) and the U:U base pairs (6:25 and 8:23) in the triple pyrimidine mismatch were included in the final rounds of structure calculation. 280 torsion angle constraints describing 258 torsion angles were used. The 40% of structures with the lowest CYANA target functions were subjected to energy minimization as described (Stoldt et al., 1999). Structures were depicted using MOLMOL (Koradi et al., 1996).
Bartels, C., Xia, T., Billeter, M., Gu¨ntert, P., and Wu¨thrich, K. (1995). The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 6, 1–10.
Yeast Three-Hybrid Assay RNA:protein interactions were analyzed using the RNA-protein Hybrid Hunter Kit (Invitrogen) according to the manufacturer’s instructions. The gene regions of CVB3 3Cpro and 3CDpro were cloned into the pYESTrp3 vector. The catalytic cysteine was substituted by a glycine to inactivate the proteinases. DNA encoding the desired RNAs were cloned into the vectors pRH5⬘ and pRH3⬘ (SenGupta et al., 1996). S. cerevisiae strain L40-ura3 was used for transformation with the protein prey plasmids, the RNA bait plasmids containing a cloverleaf or a stemloop D encoding CVB3 sequence, and pHybLex/ Zeo-MS2. Transformed colonies of S. cerevisiae strain L40-ura3 were grown on selective agar plates, transferred to filter paper, lysed, and assayed for -galactosidase activity 3–6 days after transformation. For documentation, 10–15 randomly picked colonies were mixed, grown overnight in supplemented minimal salt medium, and spotted onto selective X-gal (40 mg/l) phosphate-buffered (pH 7.0) minimal salt plates. Blue yeast colonies were scored after 5–6 days of incubation at 30⬚C.
Cai, Z., Gorin, A., Frederick, R., Ye, X., Hu, W., Majumdar, A., Kettani, A., and Patel, D.J. (1998). Solution structure of P22 transcriptional antitermination N peptide-boxB RNA complex. Nat. Struct. Biol. 5, 203–212.
Barton, D.J., O’Donnell, B.J., and Flanegan, J.B. (2001). 5⬘ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J. 20, 1439–1448. Basu, S., Rambo, R.P., Strauss-Soukup, J., Cate, J.H., FerreD’Amare, A.R., Strobel, S.A., and Doudna, J.A. (1998). A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nat. Struct. Biol. 5, 986–992. Batey, R.T., and Doudna, J.A. (2002). Structural and energetic analysis of metal ions essential to SRP signal recognition domain assembly. Biochemistry 41, 11703–11710. Blair, W.S., Parsley, T.B., Bogerd, H.P., Towner, J.S., Semler, B.L., and Cullen, B.R. (1998). Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases. RNA 4, 215–225.
Cate, J.H., Hanna, R.L., and Doudna, J.A. (1997). A magnesium ion core at the heart of a ribozyme domain. Nat. Struct. Biol. 4, 553–558. Dingley, A.J., and Grzesiek, S. (1998). Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J. Am. Chem. Soc. 120, 8293–8297. Du, Z., Yu, J., Andino, R., and James, T.L. (2003). Extending the family of UNCG-like tetraloop motifs: NMR structure of a CACG tetraloop from coxsackievirus B3. Biochemistry 15, 4373–4383. Ehrenfeld, E., and Teterina, N.L. (2002). Initiation of translation of picornavirus RNAs. In Molecular Biology of Picornaviruses, B.L. Semler and E. Wimmer, eds. (Washington, DC: ASM Press), pp. 159–170. Ennifar, E., Nikulin, A., Tishchenko, S., Serganov, A., Nevskaya, N.,
Stemloop D-Proteinase 3C Interaction 247
Garber, M., Ehresmann, B., Ehresmann, C., Nikonov, S., and Dumas, P. (2000). The crystal structure of UUCG tetraloop. J. Mol. Biol. 304, 35–42.
NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif. Cell 93, 289–299.
Gamarnik, A.V., and Andino, R. (1997). Two functional complexes formed by KH domain containing proteins with the 5⬘ noncoding region of poliovirus RNA. RNA 3, 882–892.
Leong, L.E., Walker, P.A., and Porter, A.G. (1993). Human rhinovirus14 protease 3C (3Cpro) binds specifically to the 5⬘-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J. Biol. Chem. 268, 25735– 25739.
Gorenstein, D.G. (1984). Phosphorus-31 NMR: Principles and Applications (New York, NY: Academic Press). Go¨rlach, M., Wittekind, M., Beckman, R.A., Mueller, L., and Dreyfuss, G. (1992). Interaction of the RNA-binding domain of the hnRNP C proteins with RNA. EMBO J. 11, 3289–3295. Griesinger, C., Otting, G., Wu¨thrich, K., and Ernst, R.R. (1988). CleanTOCSY for 1H spin system identification in macromolecules. J. Am. Chem. Soc. 110, 7880–7882. Gu¨ntert, P., Billeter, M., Ohlenschla¨ger, O., Brown, L.R., and Wu¨thrich, K. (1998). Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND. J. Biomol. NMR 12, 543–548. Harris, K.S., Xiang, W., Alexander, L., Lane, W.S., Paul, A.V., and Wimmer, E. (1994). Interaction of poliovirus polypeptide 3CDpro with the 5⬘ and 3⬘ termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. J. Biol. Chem. 269, 27004–27014. Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol. Cell 7, 581–591. Herrmann, T., Gu¨ntert, P., and Wu¨thrich, K. (2002). Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227. Heus, H.A., and Hilbers, C.W. (2003). Structures of non-canonical tandem base pairs in RNA helices. Review. Nucleosides Nucleotides Nucleic Acids 22, 559–571. Holbrook, S.R., Cheong, C., Tinoco, I., Jr., and Kim, S.H. (1991). Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature 353, 579–581. Hu, W., Bouaziz, S., Skripkin, E., and Kettani, A. (1999). Determination of 3J(H3⬘i,Pi⫹1) and 3J(H5⬘i/H5″i,Pi⫹1) coupling constants in 13Clabeled nucleic acids using constant time HMQC. J. Magn. Reson. 139, 181–185. Huang, H., Alexandrov, A., Chen, X., Barnes, T.W., III, Zhang, H., Dutta, K., and Pascal, S.M. (2001). Structure of an RNA hairpin from HRV-14. Biochemistry 40, 8055–8064. Jiang, L., Suri, A.K., Fiala, R., and Patel, D.J. (1997). Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex. Chem. Biol. 4, 35–50. Jiang, L., Majumdar, A., Hu, W., Jaishree, T.J., Xu, W., and Patel, D.J. (1999). Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer. Structure 7, 817–827. Johnson, V.H., and Semler, B.L. (1988). Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5⬘-noncoding regions of viral RNAs. Virology 162, 47–57. Kolk, M.H., Wijmenga, S., Heus, H.A., and Hilbers, C.W. (1998). On the NMR structure determination of a 44 nt RNA pseudoknot: assignment strategies and derivation of torsion angle constraints. J. Biomol. NMR 12, 423–433. Koradi, R., Billeter, M., and Wu¨thrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55. Lebars, I., Yoshizawa, S., Stenholm, A.R., Guittet, E., Douthwaite, S., and Fourmy, D. (2003). Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII. EMBO J. 22, 183–192. Legault, P., Jucker, F.M., and Pardi, A. (1995). Improved measurement of 13C, 31P J coupling constants in isotopically labeled RNA. FEBS Lett. 362, 156–160. Legault, P., Li, J., Mogridge, J., Kay, L.E., and Greenblatt, J. (1998).
Lescrinier, E.M.H.P., Tessari, M., van Kuppefeld, F.J.M., Melchsers, W.J.G., Hilbers, C.W., and Heuss, H.A. (2003). Structure of the pyrimidine-rich internal loop in the poliovirus 3⬘-UTR: the importance of maintaining pseudo-two-fold symmetry in RNA helices containing two adjacent non-canonical base-pairs. J. Mol. Biol. 331, 759–769. Lietzke, S.E., Barnes, C.L., Berglund, J.A., and Kundrot, C.E. (1996). The structure of an RNA dodecamer shows how tandem U-U base pairs increase the range of stable RNA structures and the diversity of recognition sites. Structure 4, 917–930. Lindauer, K., Bendic, C., and Su¨hnel, J. (1996). HBexplore: a new tool for identifying and analysing hydrogen bonding patterns in biological macromolecules. Comput. Appl. Biosci. 12, 281–289. Marino, J.P., Schwalbe, H., Glaser, S.J., and Griesinger, C. (1996). Determination of ␥ and stereospecific assignments of H5⬘protons by measurement of 2J and 3J coupling constants in uniformly 13C labeled RNA. J. Am. Chem. Soc. 118, 4388–4395. Matthews, D.A., Smith, W.W., Ferre, R.A., Condon, B., Budahazi, G., Sisson, W., Villafranca, J.E., Janson, C.A., McElroy, H.E., Gribskov, C.L., et al. (1994). Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell 77, 761–771. Mueller, L., Legault, P., and Pardi, A. (1995). Improved RNA structure determination by detection of NOE contacts to exchange-broadened amino-protons. J. Am. Chem. Soc. 117, 11043–11048. Nikonowicz, E.P., and Pardi, A. (1993). An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids. J. Mol. Biol. 232, 1141–1156. Nikonowicz, E.P., Sirr, A., Legault, P., Jucker, F.M., Baer, L.M., and Pardi, A. (1992). Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 20, 4507–4513. Parsley, T.B., Towner, J.S., Blyn, L.B., Ehrenfeld, E., and Semler, B.L. (1997). Poly (rC) binding protein 2 forms a ternary complex with the 5⬘-terminal sequences of poliovirus RNA and the viral 3CD proteinase. RNA 3, 1124–1134. Paul, A.V. (2002). Possible unifying mechanism of picornavirus genome replication. In Molecular Biology of Picornaviruses, B.L. Semler and E. Wimmer, eds. (Washington, DC: ASM Press), pp. 227–246. Proctor, D.J., Schaak, J.E., Bevilacqua, J.M., Falzone, C.J., and Bevilacqua, P.C. (2002). Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions. Biochemistry 41, 12062–12075. Santti, J., Hyypia, T., Kinnunen, L., and Salminen, M. (1999). Evidence of recombination among enteroviruses. J. Virol. 73, 8741– 8749. SenGupta, D.J., Zhang, B., Kraemer, B., Pochart, P., Fields, S., and Wickens, M. (1996). A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl. Acad. Sci. USA 93, 8496–8501. Sklenar, V., Dieckmann, T., Butcher, S.E., and Feigon, J. (1996). Through-bond correlation of imino and aromatic resonances in 13C-, 15 N-labeled RNA via heteronuclear TOCSY. J. Biomol. NMR 7, 83–87. Stoldt, M., Wo¨hnert, J., Ohlenschla¨ger, O., Go¨rlach, M., and Brown, L.R. (1999). The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition. EMBO J. 18, 6508–6521. Szyperski, T., Fernandez, C., Ono, A., Wu¨thrich, K., and Kainosho, M. (1999). The 2D [31P] spin-echo-difference constant-time [13C, 1H]HMQC experiment for simultaneous determination of 3J(H3⬘P) and 3 J(C4⬘P) in 13C-labeled nucleic acids and their protein complexes. J. Magn. Reson. 140, 491–494.
Structure 248
Tanaka, Y., Kojima, C., Yamazaki, T., Kodama, T.S., Yasuno, K., Miyashita, S., Ono, A., Kainosho, M., and Kyogoku, Y. (2000). Solution structure of an RNA duplex including a C-U base pair. Biochemistry 39, 7074–7080. Teterina, N.L., Egger, D., Bienz, K., Brown, D.M., Semler, B.L., and Ehrenfeld, E. (2001). Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis. J. Virol. 75, 3841–3850. Theimer, C.A., Finger, L.D., Trantirek, L., and Feigon, J. (2003). Mutations linked to dyskeratosis congenita cause changes in the structural equilibrium in telomerase RNA. Proc. Natl. Acad. Sci. USA 100, 449–454. Todd, S., Towner, J.S., Brown, D.M., and Semler, B.L. (1997). Replication-competent picornaviruses with complete genomic RNA 3⬘ noncoding region deletions. J. Virol. 71, 8868–8874. Varani, G., Cheong, C., and Tinoco, I., Jr. (1991). Structure of an unusually stable RNA hairpin. Biochemistry 30, 3280–3289. Walker, P.A., Leong, L.E., and Porter, A.G. (1995). Sequence and structural determinants of the interaction between the 5⬘-noncoding region of picornavirus RNA and rhinovirus protease 3C. J. Biol. Chem. 270, 14510–14516. Wo¨hnert, J., Dingley, A.J., Stoldt, M., Go¨rlach, M., Grzesiek, S., and Brown, L.R. (1999). Direct identification of NH·N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy. Nucleic Acids Res. 27, 3104–3110. Wo¨hnert, J., Go¨rlach, M., and Schwalbe, H. (2003). Triple resonance experiments for the simultaneous correlation of H5/H6 and exchangeable hydrogens of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules. J. Biomol. NMR 26, 79–83. Xiang, W., Harris, K.S., Alexander, L., and Wimmer, E. (1995). Interaction between the 5⬘-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication. J. Virol. 69, 3658–3667. Zell, R., and Stelzner, A. (1997). Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5⬘- and 3⬘-nontranslated regions. Virus Res. 51, 213–229. Zell, R., Klingel, K., Sauter, M., Fortmu¨ller, U., and Kandolf, R. (1995). Coxsackieviral proteins functionally recognize the polioviral cloverleaf structure of the 5⬘-NTR of a chimeric enterovirus RNA: influence of species-specific host cell factors on virus growth. Virus Res. 39, 87–103. Zell, R., Sidigi, K., Bucci, E., Stelzner, A., and Go¨rlach, M. (2002). Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C. RNA 8, 188–201. Zell, R., Sidigi, K., Henke, A., Schmidt-Brauns, J., Hoey, E., Martin, S., and Stelzner, A. (1999). Functional features of the bovine enterovirus 5⬘-non-translated region. J. Gen. Virol. 80, 2299–2309. Zwahlen, C., Legault, P., Vincent, S.J.F., Greenblatt, J., Konrat, R., and Kay, L.E. (1997). Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage N-peptide/boxB RNA complex. J. Am. Chem. Soc. 119, 6711–6721. Accession Numbers Coordinates have been deposited with the Protein Data Bank under ID code 1RFR and the Research Collaboratory for Structural Bioinformatics ID code 020708.
