Structural Change in Rev Responsive Element RNA of HIV-1 on Binding Rev Peptide

J. Mol. Biol. (1996) 264, 863–877

Structural Change in Rev Responsive Element RNA of HIV-1 on Binding Rev Peptide Robert D. Peterson and Juli Feigon* Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles CA 90095, USA

The HIV-1 Rev responsive element (RRE) high-affinity binding site was studied by homonuclear and heteronuclear NMR. Two Rev binding element (RBE) RNA oligonucleotides were used as model systems in this study: RBE3, which contains the wild-type Rev high-affinity binding site, and RBE3-A which is identical except for the deletion of a bulged A. The temperature dependence of the two-dimensional spectra of the free RNAs indicates that at lower temperatures more than one conformation is present. However, at higher temperatures a single conformation predominates. Model structures of RBE3 and RBE3-A as well as the RBE3-A complexed with a peptide derived from the RNA binding domain of HIV-1 Rev, were calculated using NMR-derived restraints. The Rev high-affinity binding site of the HIV-1 RRE contains a structured internal loop with two purine·purine base-pairs and an extrahelical U. Comparison of the free and bound RNA structures reveals that upon peptide binding there is a distinct change in the backbone at G24, which is involved in a G·G base-pair. In the free RNA, G24 is in the syn conformation, and the backbone is in a relatively normal configuration, antiparallel to the other strand. In the bound RNA, the backbone at G24 has flipped over so that it is parallel to the other strand. G24 in the bound RNA still forms a base-pair with G6, but is now in the anti conformation. 7 1996 Academic Press Limited

*Corresponding author

Keywords: NMR; two-dimensional NMR; RNA structure; Rev-RRE interaction; G·G base-pair

Introduction The Rev protein of human immunodeficiency virus type 1 (HIV-1) is an essential regulatory protein, and is crucial in the life cycle of HIV-1 (Feinberg et al., 1986; Sodroski et al., 1986). Rev binds to a specific region of the HIV-1 mRNA known as the Rev responsive element (RRE; Cochrane et al., 1990; Daly et al., 1989; Felber et al., 1989; Malim et al., 1989; Rosen et al., 1988; Zapp & Green, 1989), and this Rev-RRE association promotes the expression of the HIV structural proteins (Chang & Sharp, 1989; Emerman et al., 1989; Felber Abbreviations used: RRE, Rev responsive element; RBE, Rev binding element; HIV-1; human immunodeficiency virus type 1; NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single-quantum coherence; COSY; correlated spectroscopy; TOCSY, total correlation spectroscopy; RMSDs, root-mean-square deviations; HOHAHA, homonuclear-Hartmann-Hahn; HMQC, heteronuclear multiple-quantum coherence. 0022–2836/96/500863–15 $25.00/0

et al., 1989; Hammarskjo¨ld et al., 1989; Malim et al., 1989; Rosen et al., 1988). In the absence of the Rev-RRE interaction the structural proteins cannot be made, and the virus cannot replicate. Therefore the Rev protein and the RRE are potential targets for antiviral agents, and knowledge of the RRE RNA structure may be useful in designing such agents. The complete RRE is about 350 nucleotides in length and has a complex secondary structure (Kimura & Ohyama, 1994; Mann et al., 1994). Rev binds first to the RRE at a specific high-affinity binding site, and subsequently 10-11 additional Rev molecules oligomerize along adjacent lower-affinity binding sites on the RRE (Mann et al., 1994). The RRE high-affinity binding site for Rev has been localized to nucleotides 103 to 111 and 123 to 133 (RRE numbering from Mann et al., 1994) of the RRE, and consists of a ten base internal loop and surrounding stems. Several Rev binding elements (RBEs) were made using these conserved sequences (Bartel et al., 1991). In a previous NMR study (Peterson et al., 1994), RBE3, a 30 nucleotide RNA that contains the high-affinity binding site for Rev, 7 1996 Academic Press Limited

864

Structure of the Rev binding site of the HIV-1 RRE

bound forms of the RNA. However, there is a significant change in the RNA conformation when it is bound by Rev22. The sugar-phosphate backbone undergoes a local reversal at G24. G24, which is base-paired to G6, remains in place, but the ribose flips over, with the result that G24 changes from syn in the free RNA to anti in the RNA bound by Rev22. The NMR spectra of the free RNA were analyzed as a function of temperature, and we present evidence for conformational averaging at lower temperatures.

Results

Figure 1. Sequence and proposed folding of: (a) a portion of domain II of the RRE containing the high-affinity binding site of Rev; RRE numbering was derived from Mann et al. (1994). (b) RBE3; the regions referred to in the text as stem 1, internal loop and stem 2 are indicated. (c) RBE3-A; the same as RBE3 but with a deletion of A21. (d) Amino acid residue sequence of Rev22, which contains residues 34 to 50 of Rev plus four alanine residues and an arginine at the C terminus. Rev22 is succinylated at the N terminus and amidated at the C terminus.

was examined both free and in the presence of a peptide derived from the arginine-rich sequence found in Rev (Rev22; Tan et al., 1993). RBE3 was found to have a flexible but structured internal loop containing a ‘‘purine-rich bubble’’ (term coined by Heaphy et al., 1991), and structural elements which had been predicted for the RNA bound by Rev were present both in the free RBE3 and in RBE3 bound by Rev22. Battiste et al. (1994) studied a very similar RNA-Rev22 complex and identified similar structural features of the RNA internal loop including the predicted G·G and G·A base-pairs for the complex, and more recently presented an NMRbased structural model of the RNA-peptide complex (Battiste et al., 1995). However, no structures of the free RBE RNA have yet been reported. Here we present NMR-derived model structures of the free RBE3 and RBE3-A (a variant of RBE3 in which the bulged A has been deleted). These are compared to model structures of the complex formed between RBE3-A and Rev22. The molecules used in these studies are shown schematically in Figure 1. The calculated model structures show that key structural features, including the two purinepurine base-pairs and a looped-out U in the purine-rich bubble, are present in both the free and

We previously reported 1H NMR spectra and assignments on RBE3, a 30 base RNA which contains the Rev high-affinity binding site with two extra base-pairs on stem IID and the wild-type hairpin loop replaced by an extra stable UUCG tetraloop (Cheong et al., 1990), as well as preliminary results on RBE3-A (RBE3 with A21 deleted), and RBE3-AA (RBE3 with G6 and G24 replaced by As) (Peterson et al., 1994). Assignments of the base, H1', H2' and a few H3' and H4' proton resonances of RBE3 were obtained from analysis of the two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) spectra. Qualitative analysis of the NOESY spectra of RBE3 and RBE3-A indicated that the purine-rich bubble in both RNAs has the same conformation, in which the G24 is in the syn conformation and pairs with G6. Preliminary assignments of the RBE3/Rev22 complex were also presented. However, due to poor spectral quality relatively few proton assignments could be made. In order to obtain a structural model of the high-affinity binding site of Rev responsive element RNA and its complex with Rev protein and to gain an understanding of the protein-RNA recognition process, we have extended this work to a more detailed study of RBE3-A and the RBE3-A/Rev22 complex. RBE3-A gives somewhat better NMR spectra than RBE3 both free and when bound by Rev22. Removing the bulged A effectively reduces the size of the internal loop from ten to five nucleotides. It has previously been found that deletion of the bulged A21 (RBE3 numbering) does not reduce binding of RRE RNA to Rev (Heaphy et al., 1991; Kjems et al., 1992), and gel-shift experiments also indicate nanomolar binding of the Rev22 peptide to RBEs (unpublished results). In addition to the studies on RBE3-A, heteronuclear two- and three-dimensional spectra of uniformly 13C,15N-labeled RBE3 were used to check and extend the assignments of the RBE3 and RBE3/Rev22 complex, and these are compared to results obtained on RBE3-A. Exchangeable proton spectra of RBE-A Figure 2 shows a one-dimensional spectrum of the imino proton region of RBE3-A, and the

Structure of the Rev binding site of the HIV-1 RRE

Figure 2. (a) One-dimensional imino proton spectrum of RBE3-A at 1°C. The spectrum was acquired with 4096 complex points, 256 scans, and a spectral width of 10,000 Hz and apodized by exponential multiplication with a line broadening of 3 Hz. (b) Portion of a NOESY spectrum of RBE3-A in H2 O at 1°C and tm = 150 ms, showing the imino proton region. Assignments of the imino protons are shown at the top of (a). The spectrum was acquired with 2048 and 512 complex points in t1 and t2 , respectively, 64 scans per t1 increment, and a spectral width of 10,000 Hz in both dimensions and apodized with a 72°-shifted squared sine-bell in both dimensions, and zero filled to 2048 and 1024 points in v2 and v1 respectively. The sample was 4 mM RBE3-A in 10 mM phosphate (pH 6), 100 mM NaCl, and 90% H2 O/10% 2H2 O.

corresponding region of the NOESY spectrum. As in RBE3, the strong imino-imino NOE between U13 and G16 provided evidence for formation of the UUCG tetraloop (Cheong et al., 1990), indicating that the RNA is folded as expected. Sequential imino-imino NOEs are observed for the central two base-pairs of stem 1 and for all of stem 2 except the C7·G23 base-pair next to the internal loop. The additional unassigned imino resonances do not give rise to any NOEs in the two-dimensional NOESY spectrum. However, this extra imino resonance intensity provides evidence for some hydrogen bonding of the internal loop imino protons. This suggests that there is base-pairing in the internal loop, but that the internal loop imino protons are more accessible to solvent than the imino protons from the stems. RBE3-A/Rev22 complex formation Rev22 is an arginine-rich peptide which contains residues 34 to 50 of Rev, which binds the RRE with

865

Figure 3. One-dimensional imino proton spectra at 1°C of (a) RBE3-A, (b) 1:0.5 RBE3-A:Rev22, and (c) 1:1 RBE3-A:Rev22. Peaks labeled G represent either G6 or G24. Spectra were acquired with 4096 complex points, 256 scans, and a spectral width of 10,000 Hz and apodized by exponential multiplication with a line broadening of 3 Hz. RBE3-A was 4 mM (a) or 1.9 mM (b) and (c) in 10 mM phosphate (pH 6), 100 mM NaCl, and 90% H2 O/10% 2H2 O.

high affinity, plus four alanine residues and an arginine residue at the C-terminal end of the peptide which increases the propensity for a-helix formation and enhances the specificity of the peptide for the RRE (Tan et al., 1993). Figure 3 shows the one-dimensional spectrum of the imino proton region of RBE3-A as Rev22 is titrated in. As previously observed for the RBE3/Rev22 complex, a second set of imino resonances appears which are in slow exchange with those from the free RNA at less than stoichiometric amounts of peptide. In the 1:1 RBE3-A/Rev22 complex, sharp imino resonances are observed for the internal loop as well as the stem, indicating that binding of the peptide stabilizes the internal loop imino protons from exchange with water. Sequential imino-imino NOE crosspeaks are seen from G2 through both stems and the internal loop to the G16·U13 base-pair in the UUCG tetraloop (not shown). The imino proton spectra of the RBE3 and RBE3-A peptide complexes are very similar, so the assignment of the imino protons of the bound RBE3-A was partially done by comparison to bound RBE3 spectra (Peterson et al., 1994). As in the RBE3/Rev22 complex, a strong G6-G24 imino-imino NOE provides direct evidence for the symmetric G6·G24 base-pair, and a strong G5 imino to A26H2 NOE indicates the formation of a G5·A26 base-pair in the RBE3-A/Rev22 complex.

866

Figure 4. UV melting study of RBE3, free and with bound Rev22. Samples were melted in a Varian CARY 1E UV-visible spectrophotometer from 25°C to 90°C at 0.5°/minute. RBE3 was 0.2 mM in concentration in 10 mM phosphate (pH 6), 50 mM NaCl in H2 O. Rev22 was added to the RBE3 sample in an equimolar concentration for the melting study of the complex.

Figure 4 shows the optical melting curve of RBE3 and the RBE3/Rev22 complex. The free RNA is quite stable; it does not even begin to melt until 050°C and has a tm of 66°C. The melting temperature of the RBE3 increases substantially when bound by Rev peptide, to 73°C. Melting studies on RBE3-A, both free and bound by Rev22, were also done (results not shown). Exact melting temperatures could not be determined because at 90°C the absorbance at 260 nm was still increasing (no upper plateau of the melting curve could be reached). The melting temperature of the free RBE3-A is estimated to be 078°C, and the melting temperature of the RBE3-A bound to Rev22 is somewhat higher. Resonance assignments of RBE-3 and RBE3-A In our previous paper, assignments of the base, H1', H2' and a few H3' and H4' resonances of RBE3 were reported (Peterson et al., 1994). These assignments were based primarily on 1H NMR spectroscopy. In order to check the assignments made and to extend these assignments to additional sugar protons, heteronuclear two- and three-dimensional spectra were obtained on uniformly 13 C,15N-labeled RBE3. Assignments of the base and H1' resonances were confirmed by three heteronuclear experiments. A 15 N-1H long-range heteronuclear single-quantum coherence (HSQC) spectrum separated UH5 and UH6, CH5, and CH6, purine H8 and AH2 resonances into distinct spectral regions due to the chemical shift differences of their coupled nitrogen nuclei (Sklena´rˇ et al., 1994), which allowed base resonances to be identified by type. HCN (Sklena´rˇ et al., 1993a) and HCNCH (Sklena´rˇ et al., 1993b), as well as the 15N-1H long-range HSQC, provided intranucleotide H8/6 and H1' correlations, which

Structure of the Rev binding site of the HIV-1 RRE

confirmed the assignment of intranucleotide aromatic to H1' NOE crosspeaks. Although proton NMR experiments were sufficient to obtain assignments of most of the H1' and H2' resonances, most of the other sugar protons were not assignable by proton only methods due to spectral overlap. HCCH-COSY (Kay et al., 1990) and HCCH-TOCSY (Bax et al., 1990) experiments were used to obtain many additional sugar proton assignments. These experiments extended the assignments to almost all of the H3', H4' and about half of the H5', H5" resonances of RBE3, as well as to the attached carbon and nitrogen resonances. The non-exchangeable proton spectra of RBE3-A were essentially identical to those of RBE3 except for the resonances near the deleted A21. These showed only very slight chemical shift differences due to the fact that A21 is looped out of the helix in the free RBE3. Thus, assignments for RBE3-A were largely obtained from the 1H spectra, and were confirmed by comparison with RBE3. Heteronuclear spectra were also obtained on a 13 C,15N-G-labeled sample of RBE3-A, and confirmed the assignments made from the proton spectra. As previously reported, the NOESY spectra of RBE3 and RBE3-A taken in 2H2 O clearly show that the internal loops of the RBEs are structured (Peterson et al., 1994). The NMR data on both RBE3 and RBE3-A provide evidence that a Gsyn ·Ganti base-pair is present in the free RNA; i.e. G24 is syn and NOE crosspeaks which place the two Gs in the right position to form a base-pair are observed. A set of NOE crosspeaks from A26H2 to C27H1' and G6H1' also place A26 and G5 in the right position to form an Aanti ·Ganti base-pair (Peterson et al., 1994).

Proton assignments of RBE-peptide complexes Although the imino proton spectra of RBE3 and RBE3-A complexed to Rev22 show sharp exchangeable resonances, the two-dimensional non-exchangeable proton spectra of the complexes are generally broad and therefore difficult to assign. The best spectra were obtained at 20°C. It was previously reported (Peterson et al., 1994) that G24 of RBE3 in complex with Rev22 was in the syn conformation as it is in the free RNA. The original assignments of RBE3 bound to Rev22 were entirely based on proton experiments. However, a constant time 13C-1H HSQC experiment done on a complex of 13C,15N-labeled RBE3/Rev22 with the constant time set to 1/JHC shows three H1'-C1' crosspeaks that are unusually upfield shifted (Figure 5). One of these belongs to G17 from the UUCG tetraloop, and one belongs to C27, but the third is inexplicable given the previous assignments. The NOESY spectra were then reexamined, and it was found that G24H1' and A26H1' had been misassigned due to several unusual chemical shifts and to broad lines in the NOESY spectra. Figure 6 shows the correct sequential assignment pathway in a NOESY

867

Structure of the Rev binding site of the HIV-1 RRE

with short mixing or transfer times worked successfully on the RNA-peptide complexes. Due to the large number of arginine residues as well as the poor spectral quality of the complexes, the peptide resonances are as yet unassigned. Temperature dependence of NOESY spectra of RBEs

Figure 5. Portion of a constant time HSQC spectrum at 40°C of 1:1 RBE3:Rev22 with fully 13C,15N-labeled RBE3 showing the C1'-, C4'-H1' regions. The constant time delay was set to 23.16 ms (01/JHC ) so that the phase of the C1' and C5' peaks is opposite to the phase of the C2', C3' and C4' peaks. The three peaks shifted into the H4' region represent the H1'-C1' correlations of G17, C27 and G24. Note that the C1' chemical shift of the G24 correlation is also shifted into the C4' region. The spectrum was acquired with 1024 and 200 complex points in t2 and t1 , respectively, 48 scans per t1 increment, and a spectral width of 5000 Hz in t2 and 4808 Hz in t1 and apodized with a 90°-shifted squared sine-bell in both dimensions, and zero filled to 2048 and 1024 points in v2 and v1 respectively. RBE3 and Rev22 were 1.2 mM in concentration in 10 mM phosphate (pH 6), 100 mM NaCl, and 90% H2 O/10% 2H2 O.

spectrum of RBE3-A bound to Rev22 (the spectra of bound RBE3 and RBE3-A are virtually identical in this region of the molecule). The corrected assignments of RBE3/Rev22 and the assignments reported here for the RBE3-A/Rev22 complex are also in agreement with the recent report by Battiste et al. (1995) on a closely related RBE. The base, H1' and H2' resonance assignments for RBE3-A and RBE3-A/Rev22 are given in Table 1. Based on these assignments, the NOESY spectra of both complexes indicate a structure in which G24·G6 form the same type of base-pair as in the free RNA, but the G24 is in the anti conformation (discussed below). Although some heteronuclear experiments, such as the constant time 13C-1H HSQC, worked reasonably well on the RBE-peptide complexes, it is worth noting that many of the experiments which gave excellent spectra on the free RBEs did not work well for the larger RBE-peptide complexes. None of the HCN, HCNCH, or long range 15N-1H HSQC experiments gave reliable base-H1' correlations. Homonuclear NOESY spectra on the labeled samples had crosspeaks which were in general too broad to be useful. In general, as might be expected, only the heteronuclear experiments

The best NOESY spectra for RBE3 and RBE3-A were obtained at 40°C, so the 40°C spectra were used to obtain information for the assignments and structure calculations. As the temperature is lowered below 40°C, NOESY crosspeaks from residues in the internal loop broaden and/or lose intensity, although their chemical shifts remain approximately the same. The lower the temperature, the broader the internal loop crosspeaks get. At 10 to 20°C many of the peaks are missing entirely or are so broad that they cannot be assigned. In contrast to the behavior of the internal loop, resonances from the stems and UUCG tetraloop stay sharp. This temperature dependence of the NOESY spectra is indicative of some sort of conformational exchange in the internal loop which is not present in the rest of the molecule. RBE3, which has a larger internal loop than RBE3-A, shows line broadening at higher temperatures and to a greater extent than RBE3-A. In order to investigate the temperature dependence of the lineshape in the absence of NOE effects, 1H-13C HSQC spectra of 13C,15N-G-labeled RBE3-A were taken at various temperatures (Figure 7). A G-labeled rather than a fully labeled sample was used so that crosspeaks would be clearly resolved, since the aromatic and H1'-C1' regions of the HSQC spectra of fully 13C,15N-labeled RBE-A are very overlapped. Linewidths of the crosspeaks were obtained from the one-dimensional slices through the crosspeaks by linefitting. The crosspeaks from the G residues in the internal loop, i.e. G24, G5 and G6, lose intensity and broaden out as the temperature is lowered, while those from the G residues in the stem and UUCG tetraloop broaden only slightly, as expected from the increase in correlation time as the temperature is lowered. Model structure analysis Although the temperature dependence of the spectra of the RBEs indicates that there is some conformational exchange in their internal loops at lower temperatures, both the chemical shift and NOE data are consistent with a predominant conformation at 40°C. The available data are not sufficient to allow the calculation of high-resolution structures of these RNAs. However, it has been possible to calculate NMR-based model structures of both RBE3 and RBE3-A free in solution and of RBE3-A when bound by Rev22 that show the major structural features. The input restraints for the structure calculations are summarized in Table 2.

868

Structure of the Rev binding site of the HIV-1 RRE

Figure 6. Portion of a 750 MHz NOESY spectrum of 1:1 RBE3-A/ Rev22 in 2H2 O at 20°C and tm = 300 ms showing the region containing the RNA aromatic-H1', H5 crosspeaks. Assignments of the H8 and H6 resonances are indicated on the side of the spectrum. Connectivities from G23 to G24 are indicated with continuous lines and from G24 to A26 with a broken line. The spectrum was acquired with 2048 and 650 complex points in t2 and t1 , respectively, 48 scans per t1 increment, and a spectral width of 6775 Hz in both dimensions and apodized with gaussian multiplication with a line broadening of −10 and a gaussian broadening of 0.18 in both dimensions, and zero filled to 1024 points in both dimensions. RBE3-A and Rev22 were 1.9 mM in 10 mM phosphate (pH 6), and 100 mM NaCl in 2H2 O.

Superpositions of the five lowest energy structures of free RBE3 and RBE3-A are shown in Figure 8. Superpositions of the heavy atoms of nucleotides 4-7 and 23-27 (excluding U25) are shown, looking into the major groove at the purine-rich bubble. This superposition includes the purine-purine base-pairs as well as one WatsonCrick base-pair on either side. Superpositions of the five lowest energy structures of Rev22-bound RBE3-A using the same nucleotides are shown in Figure 9. Table 3 shows root-mean-square deviations (RMSDs) for several regions of the molecules, RBE3, RBE3-A and RBE3-A bound by Rev22. The overall RMSDs for these molecules are rather high and the full structures do not superimpose well, especially in the case of RBE3. However, the RMSDs for smaller regions of the molecules, for example superpositions of only stem 1, the internal loop, or stem-loop II are much lower. RBE3 has higher RMSDs than RBE3-A because of the larger internal loop which is less constrained than the stems in the calculations. Although the relatively high overall RMSDs of the molecules are likely due largely to lack of long-range constraints in general and also to the lack of backbone dihedral restraints in the internal loop, it is also possible that this represents a genuine flexibility in the molecules.

Discussion Model structures of RBE3 and RBE3-A The model structures of RBE3 and RBE3-A show the global fold and overall structural features of the

internal loops of these molecules. Both molecules show the same significant features in the purinerich bubble, i.e. G24 is in the syn conformation, U25 is looped out of the helix, and G24 and A26 are stacked on each other. Both G24 and A26 are involved in purine·purine base-pairs in the structures, i.e. G24syn ·G6anti and A26anti ·G5anti , which close the internal loop. The RNA is also underwound at the purine-rich bubble, making the major groove wider and shallower than in standard A-form duplexes. The larger internal loop in RBE3 contains additional Watson-Crick base-pairs plus the unpaired A, which is looped out of the helix. Conformational change on peptide binding The model structure of RBE3-A in complex with Rev22 is very similar to that of free RBE3-A. Both purine-purine base-pairs are present, and U25 is looped out of the helix. There is, however, one very striking change. The backbone undergoes a reversal at G24. G24 is in the syn conformation in the free RNA, and is thus flipped upside down from the usual anti position. The symmetric base-pair that G6 and G24 forms requires that one of the two be upside down relative to the normal orientation of adjacent bases along the same strand. In the RBE3-A/Rev22 complex, the base-pair still forms, and G24 is still flipped relative to G23 and A26, but G24 is anti, not syn. To accommodate this, the backbone has also undergone a reversal at G24 on Rev22 binding. Thus the entire nucleotide is turned over instead of just the base, leaving G24 anti but still turned over. The major groove at the

869

Structure of the Rev binding site of the HIV-1 RRE

Table 1. Chemical shifts (ppm) of RNA proton resonances for RBE3-A (free) and the RBE-3-A/Rev22 complex (bound) at 40°C (free), and 20°C (bound) H6/H8

5' G1 G2 U3 G4 G5 G6 C7 G8 C9 A10 G11 C12 U13 U14 C15 G16 G17 C18 U19 G20 C22 G23 G24 U25 A26 C27 A28 C29 C30

Freea

Boundb

8.13 7.59 7.70 7.66 7.70 7.67 7.75 7.60 7.66 7.97 7.28 7.41 7.78 8.06 7.72 7.88 8.32 7.69 7.86 7.74 7.54 7.38 7.72 7.89 8.10 7.55 8.06 7.52 7.64

8.09 7.54 7.60 8.14 8.61 8.00 7.71 7.59 7.60 7.93 7.26 7.35 7.74 8.01 7.69 7.86 8.34 7.74 7.73 7.66 7.45 7.12 7.83 7.99 7.95 7.46 7.90 7.23 7.56

H2/H5 Free

H1'

Bound

5.16

4.20

5.29

5.06

5.25 7.01

5.19 6.89

5.16 5.72 5.89 6.16

5.13 5.69 5.84 6.12

5.31 5.49

5.24 5.08

5.20

4.84

5.86 7.92 5.43 7.42 5.20 5.46

5.92 7.60 5.25 7.47 5.28 5.42

H2'

Free

Bound

Free

Bound

5.86 5.93 5.58 5.71 5.75 5.70 5.57 5.70 5.46 5.93 5.66 5.51 5.69 6.11 5.98 5.98

5.85 5.95 5.47 5.34 5.91 5.81 5.68 5.76 5.39 5.86 5.62 5.44 5.65 6.10 5.96 5.97

4.97 4.61

4.99 4.58 4.27

4.32 4.67 4.51 4.55 4.49 4.47 4.62 4.43 4.48 3.82 4.69 4.12 4.86

4.74 4.62 4.46 4.44 4.58 4.42 4.45 3.79 4.68 4.11 4.85

5.55 5.57 5.77 5.39 5.68 5.62 6.02 5.80 5.33 5.92 5.43 5.79

5.51 5.47 5.73 5.25 5.80 4.87 6.18 5.38 4.93 5.85 5.19 5.72

4.47 4.65 4.5 4.33 4.39 4.70 4.47 4.76 4.32 4.52 4.11 4.02

4.46 4.57 4.37 3.97 4.39 3.99 4.44 4.64 4.16 4.47 4.04 3.99

Chemical shift changes between free and bound of greater then 0.2 ppm are shown in bold. a Sample is 10 mM phosphate (pH 6), 100 mM NaCl, 4 mM RBE3-A. b Sample is 10 mM phosphate (pH 6), 100 mM NaCl, 1.9 mM RBE3-A + Rev22.

purine-rich bubble in the bound form of RBE3-A is even more underwound and wider than it is in the free form. The structural change that takes place when Rev22 binds to RBE3-A is shown in Figure 10(a) using representative structures, and schematically in Figure 10(b). One would think that the conformation that the backbone in the region of G24 and U25 adopts on binding Rev22 would require some direct contacts with the peptide for stabilization. However, no specific sites on the bases in the purine-rich bubble have been identified as being involved in direct contacts with Rev. Most functional groups on the four purine nucleosides in the bubble have been replaced using modified nucleosides (Iwai et al., 1992; Pritchard et al., 1994), and the only replacements which affected the binding to Rev are those which disrupted the base-pairing. In addition, all of the phosphate groups of the internal loop of the RRE have been analyzed by ethylation interference (Kjems et al., 1992) or methylphosphonate substitution (Pritchard et al., 1994). The ethylation interference analysis suggested only the 3' phosphate groups of G104 and G105 (G4 and G5 of RBE3) as being possible contact sites of Rev. However, the 3' phosphate group of G104, G105 and G106 were also analyzed by methylphosphonate substitution, a more sensitive technique, and it

was found that substitution of P104 and P106 affected binding to Rev. Since the structures of the sarcin/ricin and E loops of rRNA also contain a reversed nucleotide adjacent to a bulged nucleotide (Szewczak et al., 1993; Wimberly et al., 1993), it has been suggested that, in the context of the RRE, G24 anti may be more sterically favorable than G24 syn (Battiste et al., 1995). This is clearly not the case, since G24 is in the syn conformation in the free RNA. However, the free energy of the two conformations may be very close, making it unnecessary for the peptide to directly contact the backbone in the region G24 to A26. Future structural studies on the Rev-RRE complex should shed additional light on this. Function of U25 Iwai et al. (1992) proposed that in the RRE the identity of the base at position 130 (corresponding to U25 of RBE3) does not affect the binding to Rev, because Rev binding tolerates chemical modification of U130 or replacement of U130 by other nucleotides (Heaphy et al., 1991; Iwai et al., 1992; Le et al., 1990). Substitution of U130 with a three-carbon propyl linker has no effect on Rev binding to an RRE RNA (Pritchard et a 1994). This suggests that U25 acts as a spacer to position correctly the

870

Structure of the Rev binding site of the HIV-1 RRE

Figure 7. Portions of HSQC spectra at 10°, 20°, 30° and 40°C of 13 C,15N-G-labeled RBE3-A, showing the aromatic regions. H8-C8 correlations from G5 and G24, from the internal loop, and G17, a typical stem nucleotide, are indicated. The carbon pulses were centered at 0139 ppm to excite the aromatic groups, and the INEPT delay was set to 1.47 ms to maximize the H8/H6-C8/C6 transfer. The spectra were acquired with 2048 and 200 complex points in t2 and t1 , respectively, eight scans per t1 increment, and a spectral width of 6010 Hz in t2 and 2000 Hz in t1 and apodized with a 72°-shifted squared sine-bell in both dimensions, and zero filled to 2048 and 1024 points in v2 and vl respectively. RBE3-A was 1.3 mM in concentration in 10 mM phosphate (pH 6), and 100 mM NaCl in 2H2 O.

surrounding purine-purine base-pairs. Examination of the model structures of RBE3-A free and bound to Rev22 suggests that this view is essentially correct. The purine-purine base-pairs in the free RNA where G24 is syn could almost certainly form in the absence of a bulged base; however, without the extra play in the backbone introduced by an extrahelical nucleotide between G24 and A26, the backbone at G24 could not flip. This change in the backbone conformation may be necessary to position the backbone or nearby base-pairs for direct contacts to Rev. It is also possible that the extra distortion of the backbone in the free RNA caused by a bulged base (or a three-carbon linker) helps in the initial recognition of the RNA by Rev. Comparison to other model structures of RBE-peptide complexes Battiste et al. (1995) have also reported a model structure for an RBE/Rev22 complex. It is of interest to compare these two structures, since the

RNAs used are closely related but not identical. Furthermore, these two complexes provide an opportunity to assess how well it is possible to determine these structures given their relatively large size and flexibility and in the absence of additional restraints that may be provided by assigned peptide NOEs. The RNA studied by Battiste et al. (1995) differs from RBE3 in that stem 1 has six base-pairs and the hairpin loop is a GCAA loop rather than a UUCG loop. In addition, the RNA used by Battiste et al. (1995) contains the bulged A (21 of RBE3), making the sequence of the binding site of their RNA identical to that of RBE3. The same peptide was used in both studies. Structural studies done by Battiste et al. (1995) were done only on the RNA bound to the Rev peptide, and not on the free RNA. The structural features identified by Battiste et al. (1995) of the internal loop of their RNA are essentially the same as those found in this study for the bound form of RBE3-A. In both cases, the NMR data were adequate only to obtain NMR-

Table 2. Input restraints for structure calculations NOE total Exchangeable NOEs Interresidue NOEs Dihedral angle restraints

RBE3

RBE3-A

RBE3-A/bound

180 15 119 147

146 11 105 168

171 19 116 168

Structure of the Rev binding site of the HIV-1 RRE

871

(a)

(b)

Figure 8. Superpositions of the five lowest-energy RBE3 (a) and RBE3-A (b) model structures. Structures were superimposed to the lowest energy structure, and the superposition was done on residues 4 to 7, 23 to 24 and 26 to 27. This includes the two purine-purine base-pairs, and the two adjacent base-pairs, but leaves out the bulged U25. The view is looking into the major groove at the purine-rich bubble. The nucleotides in the G6·G24 and G5·A26 base-pairs are blue, U25 is red, base-pairs G4-C27 and C7-G23 are purple, and the remaining bases are green.

based model structures for the RBE/Rev peptide complexes, in the absence of the additional constraints that would be provided by complete

assignment of the peptide. Nevertheless, the models of the free RBEs reported here compared to the RBE/Rev peptide complex clearly illustrate an

872

Structure of the Rev binding site of the HIV-1 RRE

Figure 9. Superpositions of the five lowest-energy Rev22-bound RBE3-A model structures. The superposition and color scheme are the same as for Figure 8. The view is looking into the major groove at the purine-rich bubble.

important structural difference between the free and bound RNA. Conformational exchange in internal loops of RBEs At the higher temperatures the NOESY data clearly indicate that the internal loop of the free RBEs has a predominant conformation as described. However, in NOESY (not shown) and HSQC (Figure 7) spectra of RBE3-A taken at temperatures ranging from 10°C to 40°C crosspeaks from internal loop resonances lose intensity and broaden as the temperature is lowered while stem and hairpin loop resonances remain sharp and at full intensity. In the temperature range studied, no significant changes in chemical shift are seen for these resonances. The same effect is also seen in NOESY spectra of RBE3. This broadening and/or

loss of intensity indicates conformational exchange in the internal loop. One possible explanation for the spectral changes as a function of temperature is that this region of the molecule has one or more alternate conformations. For example, the alternate conformation could be the conformation the RBE adopts when bound by Rev22 (i.e. the switch of G24 from syn to anti ). At first glance this is an attractive possibility because it would make it easier to understand the binding of Rev22 to the RNA. However, this would predict that the resonances which have the largest chemical shift change between 10°C and 40°C in the free RNA would also have the largest change in chemical shift between the bound and unbound forms of the RNA. There is no such correlation. In addition, there is no obvious correlation between the resonances which have the largest chemical shift change between bound and unbound RNA

˚ ) of the 5 and (20) lowest-energy structures Table 3. Pairwise RMSD (A of the RBEs All residues except A21, U25, and tetraloop Internal loop (residues 4-7, 23, 24, 26, 27) Purine-purine base-pairs Stem 1 (residues 1-4, 27-30) Stem 2 (RBE3: residues 9-12, 17-20) (RBE3-A: residues 7-12, 17-23)

RBE3

RBE3-A

RBE3-A/bound

5.57 (4.32)

3.70 (2.83)

2.48 (2.60)

2.50 (2.56)

2.02 (1.46)

1.80 (1.67)

1.45 (1.63) 0.82 (0.68)

1.44 (1.22) 0.56 (0.38)

1.59 (1.54) 0.75 (0.66)

0.80 (0.75)

0.73 (0.82)

0.93 (1.01)

873

Structure of the Rev binding site of the HIV-1 RRE

(a)

(b)

Figure 10. (a) Representative structures of RBE3-A both free and bound by Rev22. Note the dramatic change in the backbone at positions 23 to 26. The color heme is the same as for Figure 8. (b) Schematic representation of the conformational change of RBE3-A between unbound and bound forms.

and those which broaden the most from 40°C to 10°C in the HSQC spectra of the free RNA. Finally, the calibrated integrated intensity for the G24H8H1' crosspeak indicates that it is in the syn conformation to at least 20°C. Thus it is unlikely that this possibility is correct. Another possibility is that at the lower temperatures, one or more alternative conformations exist in slow conformational exchange with the major high-temperature conformation. Examination of the sequences of the RBEs shows that alternative base-pairs could form in and around the internal loop. The observed spectral changes are consistent with this possibility, since the largest changes are observed for RBE3-AA (Peterson et al., 1994) followed by RBE3 and then RBE3-A. The internal loop of RBE3-AA has the most possibilities for alternative base-pairs while the smaller internal loop of RBE3-A has the fewest possibilities. This explanation is also consistent with the imino proton spectra, since the broad imino resonances and lack of observable NOE crosspeaks for the internal loop base-pairs could be due to exchange between alternative base-pairs as well as exchange with water. The nature of the conformational exchange remains unknown at this time. It is possibly complex, involving multiple states and changing exchange rates and/or changing populations with temperature. Experiments are currently in progress to obtain a clearer picture of this conformational exchange. What is clear is that these RNAs have a predominant conformation, at least at higher temperature, which corresponds to the model

structures calculated here, and that they are conformationally flexible. This flexibility is also demonstrated by the unusual conformational change that takes place in RBE3-A when it is bound by Rev22.

Conclusions Rev22 binds to RBE3 or RBE3-A at an internal loop which contains a G·G and a G·A base-pair and a looped-out U. The binding of Rev22 induces a conformational change in the RNA, resulting in an unusual kink in the backbone. Thus, the two purine base-pairs may provide the initial recognition determinants, while the bulged U provides the flexibility needed for the RNA to accommodate the bound peptide. The RNA is structured but conformationally flexible both free and in the complex. RNA conformational change induced by protein binding has been observed in several RNA-protein complexes (reviewed by Draper, 1995), and it seems likely that this will prove to be the general case. In most RNA-protein complexes studied to date, recognition of a non-standard duplex or loop and the ability of the RNA to adopt a different conformation on binding have been seen. Although the peptide model cannot be assumed to accurately represent the conformation of Rev protein, the conformational change in the RNA induced on peptide binding is probably an accurate reflection of the structural determinants of Rev-RRE recognition.

874

Materials and Methods Sample preparation RNA oligonucleotides RBE3 and RBE3-A were synthesized by in vitro transcription using T7 RNA polymerase by a modification of the procedures described by Milligan et al. (1987) and Wyatt et al. (1991) as previously described (Peterson et al., 1994). The T7 RNA polymerase was purified from the overproducing Escherichia coli strain UT4400/pGP1-5/pGP1-1 (a gift from Dr Stanley Tabor) or from the E. coli strain BL21/pAR1219 (a gift from the Brookhaven National Laboratory) by a modification of the procedure of Zawadzki & Gross (1991). RNA was purified by polyacrylamide gel electrophoresis under denaturing conditions followed by electroelution of the full-length band. The purified RNA was then dialyzed in Amicon Centricon filters as described (Peterson et al., 1994) or it was run on a DEAE column (50% (v/v) DEAE-sephacel, 50% DEAE-Sepharose) eluted with 1.5 M NaCl, followed by desalting on a Sephadex G15 column eluted with water. Uniformly 13C,15N-labeled RNA was enzymatically synthesized using 13C,15N-labeled NTPs which were prepared from RNA isolated from E. coli grown in 13 C-glucose- and 15N-(NH4 )2 SO4-containing media (Nikonowicz et al., 1992), or from Methylophilus methylotrophus grown in 13C-methanol and 15N-(NH4 )2 SO4 (Batey et al., 1992). Uniformly 13C,15N-G-only labeled samples were prepared as above, but using 13C,15N-labeled G and unlabeled ATP, CTP and TTP. For the selectively labeled samples, the labeled NMPs used in the synthesis were first purified by HPLC using a semi-preparative scale C18 column (Waters: PrepPak cartridge, 25 × 100 mm) with a water/methanol gradient. Peaks were identified as specific NMPs by comparison with HPLC traces of individual NMPs which were run under the same conditions. Individual labeled NMPs were then kinased separately. The peptide Rev22 was synthesized and purified as described (Peterson et al., 1994). NMR samples were prepared in H2 O (90% (v/v) H2 O/10% (v/v) 2H2 O) or 2H2 O (99.996% 2H2 O) solution with 100 mM NaCl, 10 mM sodium phosphate (pH 6.0), and 02 M RNA unless otherwise specified. RNA-Rev22 peptide complexes were prepared by titrating in the peptide as previously described (Peterson et al., 1994). The samples used in these studies were: (1) unlabeled RBE3, (2) unlabeled RBE3-A, (3) unlabeled RBE3/Rev22 complex, (4) unlabeled RBE3-A/Rev22 complex, (5) uniformly 13C,15N-labeled RBE3, (6) uniformly 13C,15Nlabeled RBE3/Rev 22 complex, and (7) uniformly 13 C,15N-G-labeled RBE3-A. NMR spectroscopy NMR spectroscopy was done at 500 MHz on GE GN500 and Bruker AMX500 and DRX500 spectrometers unless otherwise specified. A few additional spectra were obtained at 750 MHz on a Bruker DMX750 spectrometer. For each free RNA sample and RNA-peptide complex, one-dimensional spectra in H2 O were collected over a range of temperatures from 1°C to 50°C using a 11 spin-echo pulse sequence (Sklena´rˇ & Bax, 1987) to suppress the water resonance. NOESY spectra of the samples in H2 O were obtained with a 11 spin-echo read pulse sequence at 1°C to 10°C. A series of two-dimensional NMR 1H spectra was acquired at one or more temperatures for each sample in 2H2 O. These included NOESY (Kumar et al., 1980) spectra at several mixing

Structure of the Rev binding site of the HIV-1 RRE

times, P.COSY (Marion & Bax, 1988) spectra or double quantum filtered COSY (Piantini et al., 1982) spectra, and homonuclear-Hartmann-Hahn (HOHAHA; Bax & Davis, 1985) or TOCSY (Braunschweiler & Ernst, 1983) spectra at 10°, 20°, 30°, 40° and 50°C for most samples. For the 13C,15N-labeled samples, 1H-15N HSQC (Sklena´rˇ et al., 1994), and HCN (Sklena´rˇ et al., 1993a) and HCNCH (Sklena´rˇ et al., 1993b) spectra were taken to correlate intranucleotide base and sugar protons as previously described. HCCH-TOCSY (Bax et al., 1990) and constant time 1H-13C HSQC with the constant time set to 1/JH,C (Santoro & King, 1992; van de Ven & Philippens, 1992; Vuister & Bax, 1992) spectra were acquired to aid in the assignment of the sugar resonances. Three-dimensional 1 H-13C NOESY-HMQC (Marion et al., 1989) and NOESY-HSQC (Norwood et al., 1990) spectra were acquired to help in resolving NOE crosspeaks to use as distance restraints. 1 H-13C HSQC (Bodenhausen & Ruben, 1980) spectra were taken on a 13C,15N-G-labeled RBE3-A sample at several temperatures in order to study the linewidths of H8-C8 correlations with changing temperatures. These spectra were apodized with an exponential multiplication and one-dimensional slices were taken through the 1 H-13C crosspeaks in both dimensions. The linewidths of these one-dimensional slices were determined by fitting them to a Lorentzian lineshape using Igor Pro version 3.0. Data acquired on the GE GN500 spectrometer were transferred to Silicon Graphics workstations and processed with Felix (Biosym). Data acquired on the Bruker spectrometers were processed with UXNMR. Acquisition and processing parameters are given in the Figure legends. Model structure determination Interproton distance restraints were obtained from two-dimensional NOESY spectra with mixing times of 80, 150 and 300 ms at 500 MHz. For RBE3, 750 MHz NOESY spectra with mixing times of 80 and 150 ms and for RBE3-A/Rev22 a mixing time of 300 ms were used instead of the equivalent 500 MHz spectra. The NOESY spectra of the free RNA molecules were taken at 40°C and the NOESY spectra of the RBE3-A/Rev22 complex were taken at 20°C. Although three-dimensional 1H-13C NOESY-HSQC and NOESY-heteronuclear multiplequantum coherence (HMQC) spectra also were acquired for the labeled RBE3, no additional NOESY crosspeaks could reliably be identified from these spectra beyond those that were found in simple two-dimensional NOESYs due to the fast relaxation of the labeled RNA. NOE crosspeaks were introduced into structure calcu˚ ), medium (1.8 to 4.5 A ˚ ), lations as strong (1.8 to 3.5 A ˚ ), and very weak (1.8 to 7.0 A ˚ ) distance weak (1.8 to 5.5 A restraints. The non-exchangeable base protons and the 1' and 2' protons were completely assigned, and all observable NOEs from these and from assigned exchangeable protons were used as distance restraints. The remaining sugar protons were only partially assigned in 13C,15N-labeled free RBE3, and did not give rise to any additional, reliable NOEs in 13C-filtered NOESY experiments. In homonuclear experiments, the spectral region from 4 to 5 ppm, where all sugar protons except the 1' protons resonate, is so overlapped that NOE crosspeaks involving sugars other than 1' or 2' were not considered reliable. Therefore, NOEs involving 3', 4', 5', or 5" protons were not used as distance restraints in structure calculations. Hydrogen bond restraints were added for each stem

875

Structure of the Rev binding site of the HIV-1 RRE

base-pair as well as for the G·G and G·A base-pairs in the purine-rich bubble and for base-pairs C7·G23 and G8·C22 in RBE3. The in vitro selection data (Bartel et al., 1991), chemical and enzymatic footprinting data (Kjems et al., 1992; Tiley et al., 1992), and binding studies using chemically modified RNAs (Iwai et al., 1992; Pritchard et al., 1994), all indicate the formation of G·G and G·A base-pairs in the internal loop of the RRE when the RNA is bound by Rev. The NMR data on the free RNA strongly suggests that these base-pairs are also present in free RNA. In the RBE-Rev peptide complexes there is direct NMR evidence (in the form of hydrogen bonded imino protons that give rise to NOEs) for the formation of the G·G and G·A base-pairs. Therefore hydrogen bond restraints for these base-pairs were included in all model structures. Weak planarity potentials were introduced for each base-pair in the later refinement steps. Sugar puckers were constrained to be C2'-endo, or C3'-endo (as defined by Saenger, 1984), based on the size of the 1'-2' couplings (Varani & Tinoco, 1991), which were determined from P.COSY or double quantum filtered COSY experiments. Residues which had a large 1'-2' coupling (7 to 8 Hz) were constrained to be C2'-endo, and those with a small 1'-2' coupling (1 to 2 Hz or no crosspeak visible) were constrained to be C3'-endo. Residues which had an intermediate 1'-2' coupling were left unconstrained. In RBE3, residues 5, 14, 15, 21, 24 and 25 were constrained to be C2'-endo, and residues 13 and 16 were constrained to be C3'-endo. In both the free and the bound RBE3-A, residues 14, 15, 24 and 25 were constrained to be C2'-endo, and residues 13 and 16 were constrained to be C3'-endo. The stem regions of the molecules which had unambiguous C3'-endo sugar puckers and NOEs indicating A-form geometry (1 to 4, 10 to 12, 17 to 19 and 27 to 30 in RBE3, 1 to 4, 7 to 12, 17 to 23 and 27 to 30 in RBE3-A, and 1 to 3, 7 to 12, 17 to 23 and 28 to 30 in RBE3-A/Rev22) were constrained to have A-form geometry by setting the backbone dihedral angles a, b, g, d, e, z, as well as n2 to a 6° range around ideal A-form values (Saenger, 1984). Residues 7, 22 and 23 of RBE3, and residue 4 of the bound form of RBE3-A each had small to medium 1'-2' COSY crosspeaks corresponding to a 1'-2' coupling of 2 to 4 Hz. Therefore, these stem residues and their base-pairing partners were loosely restrained by setting their backbone dihedral angles to a 90° range around ideal A-form values. The x angles of all residues were loosely restrained to be in the syn (x = 60(240)°) or anti (x = −120(270)°) conformation on the basis of the H6,8-H1' NOE intensity. G16 was syn in all structures, and G24 was syn in the free RBE3 and the free RBE3-A. All other residues were anti. Structure calculations were done on Silicon Graphics Indy workstations and DEC Alpha workstations using the program X-PLOR version 3.1 (Bru¨nger, 1992). Standard X-PLOR protocols for distance geometry, template fitting, simulated annealing and refinement were used. Simulated annealing was done with 3 ps at 2000 K followed by 6 ps of cooling from 2000 K to 100 K with a step size of 0.0002 ps for RBE3 and free RBE3-A or 0.0003 ps for the bound RBE3-A. The refine protocol simulated annealing was done with 10 ps of cooling from 2000 K to 100 K with a step size of 0.001 ps. In total, 100 refined structures of each molecule were calculated from random coordinates. The convergence rate for these structures was very high, and virtually all structures had reasonable energies. The 20 lowest-energy structures were then further refined using the X-PLOR gentle refine protocol. The full van der Waals potential, including attractive forces, was turned on for the gentle refinement,

and simulated annealing was done with 10 ps of cooling from 300 K to 100 K with a step size of 0.0005 ps. All distance and dihedral restraints were included in all steps of the structure calculations. Planarity potentials for flattening the base-pairs were introduced only in the refine and gentle refine steps.

Acknowledgements We thank Dr Suzanna Horvath for supplying the peptide Rev22. This work was supported by NIH grant P01 GM39558 (J.F.) and NIH predoctoral training grant GM07185 (R.D.P.). We also acknowledge the Stable Isotope facility at Los Alamos for labeled cells and the National NMR Facility at Madison for 750 MHz NMR spectrometer time.

References Bartel, D. P., Zapp, M. L., Green, M. R. & Szostak, J. W. (1991). HIV-1 Rev regulation involves recognition of non-Watson-Crick base-pairs in viral RNA. Cell, 67, 529–536. Batey, R. T., Inada, M., Kujawinski, E., Puglisi, J. D. & Williamson, J. R. (1992). Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucl. Acids Res. 20, 4515–4523. Battiste, J. L., Tan, R., Frankel, A. D. & Williamson, J. R. (1994). Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base-pairs. Biochemistry, 33, 2741– 2747. Battiste, J. L., Tan, R., Frankel, A. D. & Williamson, J. R. (1995). Assignment and modeling of the Rev responsive element RNA bound to a Rev peptide using 13C-heteronuclear NMR. J. Biomol. NMR, 6, 375–389. Bax, A. & Davis, D. G. (1985). MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355–360. Bax, A., Clore, G. M. & Gronenborn, A. M. (1990). 1H-1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J. Magn. Reson. 88, 425–431. Bodenhausen, G. & Ruben, D. (1980). Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Letters, 69, 185–189. Braunschweiler, L. & Ernst, R. R. (1983). Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J. Magn. Reson. 53, 521–528. Bru¨nger, A. T. (1992). X-PLOR (Version 3.1) Manual, Yale University Press, New Haven and London. Chang, D. D. & Sharp, P. A. (1989). Regulation by HIV Rev depends upon recognition of splice sites. Cell, 59, 789–795. Cheong, C., Varani, G., & Tinoco, I., Jr (1990). Solution structure of an unusually stable RNA hairpin, 5'GGAC(UUCG)GUCC. Nature, 346, 680–682. Cochrane, A. E., Chen, C.-H. & Rosen, C. A. (1990). Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA. Proc. Natl Acad. Sci. USA, 87, 1198– 1202.

876 Daly, T. J., Cook, K. S., Gray, G. S., Malone, T. E. & Rusche, J. R. (1989). Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro. Nature, 342, 816–819. Draper, D. E. (1995). Protein-RNA recognition. Annu. Rev. Biochem. 64, 593–620. Emerman, M., Vazeux, R. & Peden, K. (1989). The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell, 57, 1155–1165. Feinberg, M. B., Jarrett, R. F., Aldovini, A., Gallo, R. C. & Wong-Staal, F. (1986). HTLV-III expression and production involve complex regulation at the level of splicing and translation of viral RNA. Cell, 46, 807–817. Felber, B. K., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T. & Pavlakis, G. N. (1989). Rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc. Natl Acad. Sci. USA, 865, 1495–1499. Hammarskjo¨ld, M.-L., Heimer, J., Hammarskjo¨ld, B., Sangwan, I., Albert, L. & Rekosh, D. (1989). Regulation of human immunodeficiency virus env expression by the rev gene product. J. Virol. 63, 1959–1966. Heaphy, S., Finch, J. T., Gait, M. J., Karn, J. & Singh, M. (1991). Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich ‘‘bubble’’ located within the rev-responsive region of viral mRNAs. Proc. Natl Acad. Sci. USA, 88, 7366–7370. Iwai, S., Pritchard, C., Mann, D. M., Karn, J. & Gait, M. J. (1992). Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein. Nucl. Acids Res. 20, 6465–6472. Kay, L. E., Ikura, M. & Bax, A. (1990). Proton-proton correlation via carbon-carbon couplings: a three-dimensional NMR approach for the assignment of aliphatic resonances in proteins labeled with carbon-13. J. Am. Chem. Soc. 112, 888–889. Kimura, T. & Ohyama, A. (1994). Interaction with the Rev response element along an extended stem I duplex structure is required to complete human immunodeficiency virus type 1 rev-mediated transactivation in vivo. J. Biochem. 115, 945–952. Kjems, J. R., Calnan, B. J., Frankel, A. D. & Sharp, P. (1992). Specific binding of a basic peptide from HIV-1 rev. EMBO J. 11, 1119–1129. Kumar, A., Ernst, R. R. & Wu¨thrich, K. (1980). A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem. Biophys. Res. Commun. 95, 1–6. Le, S.-Y., Malim, M. H., Cullen, B. R. & Maizel, J. V. (1990). A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses. Nucl. Acids Res. 18, 1613–1623. Malim, M. H., Hauber, J., Le, S.-Y., Maizel, J. V. & Cullen, B. R. (1989). The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature, 388, 254–257. Mann, D. A., Mikae´lian, I., Zemmel, R. W., Green, S. M., Lowe, A. D., Kimura, T., Singh, M., Butler, G., Gait, M. & Karn, J. (1994). A molecular rheostat.

Structure of the Rev binding site of the HIV-1 RRE

Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression. J. Mol. Biol. 241, 193–207. Marion, D. & Bax, A. (1988). P.COSY, a sensitive alternative for double-quantum-filtered COSY. J. Magn. Reson. 80, 528–533. Marion, D., Kay, L. E., Sparks, S. W., Torchia, D. A. & Bax, A. (1989). Three-dimensional heteronuclear NMR of 15N-labeled proteins. J. Am. Chem. Soc. 111, 1515–1517. Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, O. C. (1987). Oligoribonucleotide synthesis using T7-RNA polymerase and synthetic DNA templates. Nucl. Acids Res. 15, 8783–8798. Nikonowicz, E. P., Sirr, A., Legault, P., Jucker, F. M., Baer, L. M. & Pardi, A. (1992). Preparation of C-13 and N-15 labelled RNAs for heteronuclear multidimensional NMR studies. Nucl. Acids Res. 20, 4507–4513. Norwood, T. J., Boyd, J., Heritage, J. E., Soffe, N. & Campbell, I. D. (1990). Comparison of techniques for H-1-detected heteronuclear H-1-N-15 spectroscopy. J. Magn. Reson. 87, 488–501. Peterson, R. D., Bartel, D. P., Szostak, J. W., Horvath, S. J. & Feigon, J. (1994). 1H NMR studies of the high-affinity Rev binding site of the Rev responsive element of HIV-1 mRNA: base-pairing in the core binding element. Biochemistry, 33, 5357– 5366. Piantini, U., So rensen, O. W. & Ernst, R. R. (1982). Multiple quantum filters for elucidating NMR coupling networks. J. Am. Chem. Soc. 104, 6800– 6801. Pritchard, C. E., Grasby, J. A., Hamy, F., Zacharek, A. M., Singh, M., Karn, J. & Gait, M. J. (1994). Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins. Nucl. Acids Res. 22, 2592–2600. Rosen, C. A., Terwilliger, E., Dayton, A., Sodroski, J. G. & Haseltine, W. A. (1988). Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc. Natl Acad. Sci. USA, 85, 2071–2075. Saenger, W. (1984). Principles of Nucleic Acid Structure, Springer-Verlag, New York. Santoro, J. & King, G. (1992). A constant-time 2d overbodenhausen experiment for inverse correlation of isotopically enriched species. J. Magn. Reson. 97, 202–207. Sklena´rˇ, V. & Bax, A. (1987). A new water suppression technique for generating pure-phase spectra with equal excitation over a wide bandwidth. J. Magn. Reson. 75, 378–383. Sklena´rˇ, V., Peterson, R. D., Rejante, M. R. & Feigon, J. (1993a). Two and three-dimensional HCN experiments for correlating base and sugar resonances in 15 N, 13C labeled RNA oligonucleotides. J. Biomol. NMR, 3, 721–727. Sklena´rˇ, V., Peterson, R. D., Rejante, M. R., Wang, E. & Feigon, J. (1993b). Two-dimensional, triple-resonance HCNCH experiment for direct correlation of ribose H1', and base H8, H6 protons in 13C, 15 N-labeled RNA oligonucleotides. J. Am. Chem. Soc. 115, 12181–12182. Sklena´rˇ, V., Peterson, R. D., Rejante, M. R. & Feigon, J. (1994). Correlation of nucleotide base and sugar protons in 15N labeled HIV RNA oligonucleotide by

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Structure of the Rev binding site of the HIV-1 RRE

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Edited by I. Tinoco (Received 9 July 1996; received in revised form 26 September 1996; accepted 26 September 1996)

Note added in proof: A recent NMR structure of an RBE complexed to Rev22 (Battiste et al. (1996). Science, 273, 1547–1551) using 13C, 15N-labeled peptide, shows specific contacts between some arginine side-chains and the looped out U (U25) and the reversed G (G24) ribose. Thus the change in backbone conformation appears to be stabilized by direct peptide contacts.