Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S151

doi:10.1006/jmbi.2000.4354 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 305, 785±803

Role of Conserved Nucleotides in Building the 16 S rRNA Binding Site for Ribosomal Protein S15 Alexander Serganov1,3, Lionel BeÂnard2, Claude Portier2, Eric Ennifar1, Maria Garber3, Bernard Ehresmann1 and Chantal Ehresmann1* 1

UPR 9002 du CNRS, Institut de Biologie MoleÂculaire et Cellulaire, 15 rue Rene Descartes, 67084 Strasbourg cedex, France 2 Institut de Biologie PhysicoChimique, 13 rue Pierre et Marie Curie, 75005 Paris France 3

Institute of Protein Research Pushchino, Moscow Region 142292, Russia

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding af®nity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate ¯exibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly. # 2001 Academic Press

*Corresponding author

Keywords: rRNA; ribosomal protein S15; RNA-protein interaction; phylogenetic conservation; structure

Introduction

Present address: A. Serganov, Laboratory of Nucleic Acid and Protein Structures, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Abbreviations used: AMV, avian myeloblastosis virus; BIV, bovine immunode®ciency virus; CMCT, 1cyclohexyl-3(2-(1-methylmorpholino)ethyl)-carbodiimide; DEPC, diethylpyrocarbonate; DMS, dimethylsulfate; HIV, human immunode®ciency virus; HTLV, human T cell leukemia virus. E-mail address of the corresponding author: [email protected] 0022-2836/01/040785±19 $35.00/0

Ribosomes are built from a complex assembly of RNAs and proteins that have co-evolved to produce the contemporary molecular machine that synthesizes proteins with a high degree of ef®ciency and accuracy. According to the RNA world hypothesis, the original ribosome was probably made solely by RNA and evolved from functionally independent pieces (Noller, 1993). This is con®rmed by the recent ®nding that both the catalytic site of the 50 S subunit and the decoding site of the 30 S subunit are constituted entirely of RNA (Nissen et al., 2000; Schluenzen et al., 2000). Because rRNA molecules share sequence homologies # 2001 Academic Press

786 among all kingdoms of life, the most conserved nucleotides must represent structurally or/and functionally important regions. Present day ribosomes represent a paradigm of various RNA/ RNA, RNA/protein and protein/protein interactions. An important breakthrough is now provided by the crystallographic study of the 70 S ribosome (Cate et al., 1999), and its isolated subunits (Ban et al., 1999, 2000; Clemons et al., 1999; Schluenzen et al., 2000; Tocilj et al., 1999). Likewise, atomic-resolution details of isolated ribosomal components are becoming available from X-ray crystallography and NMR studies, allowing their placement into the structure of ribosomal subunits. Moreover, the structure of ribosomal proteins bound to their rRNA targets provides molecular insights into the recognition mechanisms between these molecules. This includes complexes involving L11 (Conn et al., 1999; Wimberley et al., 1999), L25 (Lu & Steitz, 2000; Stoldt et al., 1999), and S15 either alone (Nikulin et al., 2000) or in association with proteins S6 and S18 (Agalarov et al., 2000). Assembly of ribosomal subunits is a cooperative and ordered process initiated by the binding of the primary proteins to their rRNA targets. Among these proteins, S15 occupies a pivotal position, in relation to its ability to bind three different RNAs. First, it facilitates subsequent binding of ribosomal proteins S6, S18, S11, and S21 to 16 S rRNA (Agalarov et al., 2000; Agalarov & Williamson, 2000; Gregory et al., 1984; Held et al., 1974). The 16 S rRNA central domain and its associated proteins (S6, S8, S11, S15 and S18) build the platform, a functionally important part of the 30 S subunit, involved in ribosomal subunit association (Lee et al., 1997; Merryman et al., 1999) and placement of P-site tRNA (Moazed & Noller, 1990). Second, S15 binds to the 715 loop of 23 S rRNA, most likely serving as a ``bridge'' between the two subunits (Cate et al., 1999; Culver et al., 1999). Third, S15 binds to a pseudoknot structure on its own mRNA, just upstream of the ribosome loading site. This interaction takes place when S15 is in excess of that required for ribosome assembly and results in the repression of its own synthesis by trapping the ribosome on its loading site (Ehresmann et al., 1995; Philippe et al., 1990, 1993; Portier et al., 1990). The 16 S rRNA-S15 complex is probably one of the best biochemically studied RNA-protein complexes in ribosome. After its localization in the central domain of 16 S rRNA (Gregory et al., 1984; Ungewickell et al., 1975; Zimmermann et al., 1975), the 16 S rRNA binding site of Escherichia coli S15 (EcS15) has been characterized by footprinting techniques (Mougel et al., 1988; Powers & Noller, 1995; Svensson et al., 1988), and a minimum binding site was de®ned in closely related species (Batey & Williamson, 1996a; Serganov et al., 1996). It became evident that S15 recognized two distinct sites on 16 S rRNA. Further investigations of S15 interaction on 16 S rRNA in thermophiles, Bacillus stearothermophilus (Batey & Williamson, 1996a,b) and Thermus thermophilus (Serganov et al., 1996),

The rRNA Binding Site of Ribosomal Protein S15

have indicated that site 1 is essential for binding, while site 2 is necessary for optimal stability. Ê During the course of the present work, the 2.8 A crystallographic structure of T. thermophilus S15 (TthS15) bound to a 57 nucleotide homologous 16 S rRNA fragment containing shortened helices H20, H21 and H22 (see Figure 1) was solved (Nikulin et al., 2000), as well as the crystal structure of a larger rRNA fragment associated with proteins S6, S15 and S18 (Agalarov et al., 2000). TthS15 interacts with the expected two rRNA sites through the minor groove, without signi®cant change relative to its free conformation determined by X-ray crystallography (Clemons et al., 1998) and NMR (Berglund et al., 1997). Rather surprising, only ®ve of the 19 positively charged amino acid residues are engaged in RNA contacts, while polar residues are predominantly used. A ribbon representation of the two molecules with interacting residues is shown in Figure 2. The present work is a thorough mutagenesis investigation of the E. coli S15 RNA binding site. The effect of mutations on the RNA binding af®nity was studied by ®lter-binding assays. To better understand the effect of mutations, alterations of the RNA conformation possibly induced by mutations were probed by enzymes and chemicals in several representative variants. Finally, the contacts between EcS15 and the selected RNA variants were checked by hydroxyl radical and RNase V1 footprinting. This analysis provides insights into the role of conserved nucleotides, the relative contribution of the two binding sites and their functional relation.

Results Molecular basis of the RNA/protein interactions The RNA-binding site of protein S15 has been remarkably conserved through evolution. In particular, most nucleotides that build sites 1 and 2 are almost invariant (Figure 1(a)). E. coli and T. thermophilus RNAs differ by only the two adjacent base-pairs 656-750 and 657-749, the bulged residue at position 653, and base-pairs separating sites 1 and 2 (Figure 1(a)). On the other hand, most of the amino acid residues of TthS15 that make contact with T. thermophilus 16 S rRNA are conserved in E. coli (Figure 1(b)), with the only exception of helix a1, the less conserved part of S15. Most likely, very similar determinants are involved in both complexes, as supported by the fact that S15 from the two species bind homologous and heterologous RNAs with similar Kd (2 nM) (Serganov et al., 1996, 1997). Thus, the crystal structure of the T. thermophilus complex (Agalarov et al., 2000; Nikulin et al., 2000) can be used as a reference to get insight into the molecular details of the EcS15-16 S rRNA interactions. Site 1 includes parts of the three-way junction between helices H20, H21 and H23, and the lower part of helix H22 (Figure 2(b)). The three-way junc-

The rRNA Binding Site of Ribosomal Protein S15

787

Figure 1. The component of the S15/rRNA complex. (a) The 16 S rRNA S15 binding site. Only nucleotides from the minimum binding site de®ned by Serganov et al. (1996) are shown. The E. coli sequence is indicated, with the corresponding changes in T. thermophilus shown in small capitals. Bases in red and green are >95 % and 75 % conserved in 6000 prokaryotic sequences, respectively. (b) The E. coli S15 (EcS15) (Portier et al., 1990) compared to the T. thermophilus sequence (TthS15) (Berglund et al., 1997), with only changes indicated. Green and red residues are conserved among 23 bacterial sequences. Those additionally conserved among 55 homologous seqences from plastids, Archaea and Eukarya are shown in red. Residues found to contact rRNA in the T. thermophilus crystallographic complex (Nikulin et al., 2000) are denoted by dots.

tion was found to be constrained by a C754G654
G752 base triple and cross-strand stacking interactions between G654, A753 and G588. Nucleotides that comprise this junction are extremely conserved. For instance, nucleotides forming the C-G
G base triple are conserved in 99.5 % of prokaryotic 16 S rRNAs. The conformation of the junction is locked into place by magnesium ions and by the protein (Nikulin et al., 2000) (Figure 2(b)). The role of magnesium in triggering conformational changes of the junction is well documented (Batey & Williamson, 1998; Orr et al., 1998; Serganov et al., 1996), and the most likely explanation is that magnesium stabilizes the junction in a conformation that displays a structural complementarity with S15. Strikingly, crystallographic data unambiguously indicated that the almost invariant nucleotides that comprise the three-way junction do not provide speci®c sequence determinants, but rather trigger a unique tertiary fold of the RNA backbone that is recognized by S15. The particular bent conformation of the backbone from nucleotides 752 to 755 is recognized by amino acid side-chain located in helix a3 (Figures 2 and 3(a)). Surprisingly, among the four residues of TthS15 involved in these interactions (Arg64, Tyr68, Arg71 and Glu72), only two are conserved in EcS15 (Tyr68 and Arg71) while Arg64 and Glu72 are both replaced by lysine (Figures 1(b) and 2(a)). To better

understand how these changes are accommodated in the E. coli complex, we introduced amino acid and base changes corresponding to the E. coli RNA and protein sequences into the crystallographic structure of the T. thermophilus complex (Nikulin et al., 2000). Interestingly, the replacement by a positively charged Lys of the negatively charged Glu72, allows a direct interaction with the phosphate group of A753 (Figure 3(b)), instead of the magnesium-mediated contact in the T. thermophilus complex (Figure 3 (a)). This compensating behavior can be paralleled with the proposed recognition model of RNAs by aminoglycoside antibiotics, in which positively charged groups of aminoglycosides are able to displace magnesium ions from negatively charged pockets of RNAs (Tor et al., 1998). Otherwise, Lys64, which is substituted for Arg, is still able to contact the phosphate group of G755 (Figure 3(b)), and Arg16 in loop 1 is found at the correct distance to compensate the loss of magnesium-mediated interaction with the phosphate group of G752, caused by the Glu72 to Lys72 change (not shown). The other contacts involving Tyr68 and Arg71 and the phosphate groups of A753 and C754 were maintained (Figure 3(b)). Thus, despite the amino acid changes, compensating interactions could be formed that maintain a similar pattern of recognition, without changing the geometry of the protein backbone.

788

The rRNA Binding Site of Ribosomal Protein S15

Figure 2. Interacting amino acid and nucleotide residues in the T. thermophilus S15-rRNA complex (adapted from Nikulin et al., 2000). (a) S15 protein: the skeleton is shown in gray with a-helices and connecting loops indicated, contacting amino acids are represented by spheres and are colored according to their conservation, as in Figure 1(b). Amino acids that are different in E. coli compared to T. thermophilus are indicated. (b) The 16 S rRNA fragment. The ribose-phosphate skeleton is shown as a gray ribbon. Contacting nucleotides are indicated. Highly conserved nucleotides are in red, as in Figure 1(a). The scheme discriminates ribose-phosphate interactions (denoted by spheres) from base-speci®c recognition (highlighting the concerned base ring). Topologically related residues are boxed.

The shallow groove of helix H22 (655-657/749751) is recognized by amino acid residues located in loop 1 between helices a1 and a2, and the

N-terminal part of a2 (Figure 2). All the concerned residues (Asp20, Thr21, Gly22, Thr24 and Gln27) are conserved in both EcS15 and TthS15

Figure 3. Recognition of invariant nucleotides 752-754 by moderately conserved amino acids in T. thermophilus and E. coli. (a) Recognition of the bent backbone (752754) in the crystallographic structure of T. thermophilus. (b) Proposed recognition of the bent backbone (752-754) in E. coli.

The rRNA Binding Site of Ribosomal Protein S15

(Figures 1(b) and 2(a)). Because G656-C750/U657A749 in E. coli are substituted by C656-G750/ G657-C749 in T. thermophilus, we have tested by modeling how the concerned unchanged residues can match the E. coli RNA sequence. We found that Gln27, which hydrogen bonds with G750-N2 in the T. thermophilus complex (Figure 4(a)), is able to form an analogous bond with G656-N2 in E. coli (Figure 4(b)). The carboxyl group of Gly22, which interacts with G750-N2 in the T. thermophilus complex, can interact with G656-N2 in E. coli. Thr21 can contact A749-N3 in E. coli instead of G657-N2 in T. thermpohilus. Besides, all interactions with O20 groups of the backbone can be maintained. In addition, three residues in helix a1 (Lys4, Lys7 and Gln8) were found to interact with phosphate groups of nucleotides 658 to 660 of T. thermophilus RNA (Nikulin et al., 2000) (Figure 2). However, none of the concerned amino acid residues is conserved (Figure 1(b)). Compensating interactions are assumed to occur in the E. coli complex, but modeling is too speculative at that stage. The second site consists of a G-U/G-C motif. The G-U pair is conserved in 82 % of prokaryotic 16 S rRNAs, while G-C is found in 14 % of the sequences. The adjacent G-C is almost invariant (97 %). Highly conserved amino acid residues located in, and in the proximity of, loop 2 (His41, Asp48 and Ser51) make speci®c contacts with functional groups of the four bases in the shallow groove (Figures 2 and 3(d) of Nikulin et al., 2000). Contacts are made with the RNA backbone on adjacent nucleotides (668 and 741) by His45 and Arg34. Because the amino acid residues involved in the various interactions with 16 S rRNA are unchanged in EcS15, with the single exception of Arg34 which is replaced by Gln, the recognition of this motif is expected to be identical within E. coli and T. thermophilus complexes. It is noteworthy

789 that an identical motif is recognized in the mRNA regulatory region in a highly base-speci®c way (BeÂnard et al., 1998). The wild-type complex and target mutations In this study, we used an E. coli 16 S rRNA fragment (RNA 584-757) corresponding to a part of the central domain and containing the binding sites for proteins S6, S8, S15 and S18. This fragment is closed by the ®rst base-pairs of helix H20 and contains a 30 unpaired terminus used to anneal the primer for reverse transcription. It was veri®ed that the presence of the unpaired 30 terminus had no signi®cant effect on binding. EcS15 was prepared under native conditions from an overproducing strain. This protein bound the E. coli 16 S rRNA fragment with an apparent dissociation constant (Kd) of 1.4 nM, in good agreement with previously published data for the protein puri®ed from ribosomes (Serganov et al., 1997). The conformation of free RNA 584-767 was investigated by probing Watson-Crick positions of the four bases and N7 position of adenine with the base-speci®c probes 1-cyclohexyl-3(2(1-methylmorpholino)ethyl)-carbodiimide (CMCT), dimethylsulfate (DMS) and diethylpyrocarbonate (DEPC). The reactivity data (summarized in Table 1 and Figure 5) are similar to those published for E. coli (Mougel et al., 1988) and T. thermophilus (Serganov et al., 1996) RNAs, and are in full agreement with the crystal structure (see the following sections for details). The hydroxyl radical footprint of S15 clearly revealed the two distinct binding sites (Figures 5(a) and 6), as previously shown in both E. coli (Powers & Noller, 1995) and T. thermophilus (Serganov et al., 1996) complexes. Furthermore, there is an excellent correlation between the protected residues and those identi®ed as contact-

Figure 4. Recognition through the shallow groove of the moderately conserved 656-751/657-749 by conserved amino acids. (a) Recognition of C656-G750/G657-C749 in the crystal structure of T. thermophilus. (b) Proposed recognition of G656-C750/U657-A749 in E. coli.

790

The rRNA Binding Site of Ribosomal Protein S15

Figure 5. Summary of probing and footprinting experiments on the wild-type rRNA. The secondary and tertiary structure elements of the RNA fragment shown here take into account the crystallographic structures of the S15-rRNA complexes (Nikulin et al., 2000; Agalarov et al., 2000). In particular, the lower three-way junction shows stacking between H21 and H22, and the upper three-way junction adopts the conformation depicted in the crystallographic complex in the presence of S6 and S18 (with H23b stacking on H22 and H23a folding down parallel with H22), (Agalarov et al., 2000). (a) Reactivity of the naked rRNA at WatsonCrick positions to DMS and CMCT, and hydroxyl radical footprint of EcS15. (b) Reactivity of the naked rRNA at A(N7) to DEPC, and RNase V1 accessibility in the absence and presence of EcS15. The code is indicated in the inset.

ing ThS15 in the crystallographic structure of the T. thermophilus complex (Agalarov et al., 2000; Nikulin et al., 2000). In addition, a weak hydroxyl radical footprint was observed outside of the minimum S15 binding site, in the GAAG tetraloop of the small 23a hairpin (Figure 5(a)). It should re¯ect the additional contacts of this tetraloop with TthS15 (His50 and Arg53) that occurred in the complex containing larger rRNA and proteins S6 and S18 in addition to S15 (Agalarov et al., 2000). It is noteworthy that these contacts are not essential for S15 binding, since the upper junction and helix H23 can be deleted without signi®cant loss of af®nity (Batey & Williamson, 1996a; Serganov et al., 1996). Rather, they stabilize a tertiary conformation

that builds the S6 and S18 binding sites, subsequent to S15 binding (Agalarov et al., 2000). RNase V1 was also used to test the accessibility of double-stranded regions in the absence and the presence of EcS15. As expected, the RNase V1 footprint overlapped the hydroxyl radical footprint, yielding a much larger footprint than the hydroxyl radicals (Figures 5(b) and 7), due to the steric hindrance resulting from the large size of the probe. In addition to direct protection by the protein, RNase V1 accessibility re¯ected conformational changes triggered by S15 binding. In particular, S15 binding increased cleavage in helix H22, at positions 661 and 662 in the immediate proximity of the purine loop (Figure 7). These changes can be paralleled with the important widening of the

791

The rRNA Binding Site of Ribosomal Protein S15 Table 1. Reactivity of nucleotides to chemical probes (DMS, CMCT and DEPC) in the naked RNAs

Mutated nucleotides are indicated in red. The strength of reactivity was estimated by visual inspection from 1 to 4 (weak to very strong). To facilitate reading, the following color code was adopted: white, not determined; blue, unreactive; yellow; level 1; orange, level 2; red, levels 3 and 4. In the case of adenine, the ®rst and second numbers refer to positions N1 and N7, respectively. Values are indicated only when they differ from wild-type RNA. The appearance of strong reverse transcription pauses is indicated by (p), or (pp) for very strong pauses.

deep groove that has been observed in the S15rRNA complex in this area (Nikulin et al., 2000). In addition, a strong reduction of cleavages was observed in helix H21 and in the upper three-way junction, between helices H23a and H23b (Figure 5(b)). The latter, which can hardly be explained by direct shielding of the protein, should account for the S15-induced rearrangements postulated to provide subsequent binding sites for S6 and S18 (Agalarov et al., 2000). Similar S15-induced changes were observed in the T. thermophilus complex (Serganov, et al. 1996). In this study, we focussed on conserved nucleotides that comprise sites 1 and 2 of S15, as well as nucleotides not directly involved in binding, that might interfere with the recognition mechanism

(the bulged nucleotide on the 30 strand of helix H22 and the conserved purine-rich interior loop). Nucleotides boxed in Figure 1(a) were mutated individually or in combination with others. For each RNA variant, the apparent Kd was estimated by direct titration of 32P-labeled RNA, and in some cases by competition with wild-type RNA, using ®lter-binding assays (Figure 8). The results are given in Tables 2 and 3. In addition, possible conformational changes of selected RNA mutants were investigated by probing with chemicals (summarized in Table 1). The footprint of S15 was analyzed by hydroxyl radical and RNase V1 cleavages. Representative gels are shown in Figures 6 and 7, and the results are summarized in Tables 2 and 3.

792

The rRNA Binding Site of Ribosomal Protein S15

Figure 6. Results of hydroxyl radical hydrolysis of different RNA fragments in the presence (‡) and in the absence ( ) of EcS15. The S15-induced protections are indicated by arrowheads, ®lled for strong protection, gray for moderate and empty for weak. The two sites are indicated by bars and the concerned nucleotides are shadowed. Top, long migration; bottom, short migration.

Site 1 is crucial for S15 binding The C754-G654
G752 base triple According to the model, C754 and G654, which form a Watson-Crick pair, were unreactive at Watson-Crick positions (Table 1). The third base, G752, which interacts with the Hoogsteen side of the G-C pair (its N3 position making contact with N4 of C754, and its N2 with O6 and N7 of G654) was marginally reactive at N1. Any substitution at any of these three nucleotides was deleterious for S15 binding (Table 2). All these changes probably induced dramatic rearrangements in the geometry of the junction. As an example, the G654C substitution, which decreased the af®nity by more than two orders of magnitude (Figure 8(b)), probably led to the formation of a canonical C654-G752 pair,

as supported by the non-reactivity of C654 and G752 (Table 1). The partial reactivity of U751 was decreased, probably resulting from a stabilization effect due to the formation of a regular canonical helix. Another consequence of the formation of a C654-G752 pair was to impair the formation of the base triple with C754. The latter remaining unreactive, it might form a canonical pair with G587. Unexpectedly, U653, which was highly reactive to CMCT in the wild-type RNA, became totally unreactive (Table 1). These changes suggest a reorganization of the junction, possibly with U653 ¯ipped inside the helix instead of outside. A similar behavior was observed for the G654U substitution, with a less pronounced effect at the level of U653, the reactivity of which was decreased but not completely abolished (Table 1). A reduction of the reactivity of U653 occurred when G752 was

793

The rRNA Binding Site of Ribosomal Protein S15 Table 2. Effect of mutations on the af®nity of the rRNA for EcS15 (site 1)

The Table is divided into several sections (as in the text) with the wild-type sequence indicated (gray line). The mutated motif is shown in the ®rst column, with the mutated nucleotide in bold character, and the mutations are indicated in the second column. Krel indicates the relative binding strength. It is expressed as the ratio of the apparent dissociation constant (Kd) measured for wild-type RNA to the Kd measured for the mutant. Relative binding strength values obtained from direct saturation curves (left) or in some cases from competition assays (right). A relative Kd > 300 indicated an absence of speci®c binding, since unspeci®c binding could be observed in the presence of excess S15 without reaching a plateau (see binding of 5 S rRNA in Figure 8(a)). The ef®ciency of binding at the two sites is estimated by the extent of hydroxyl radical protection: ‡ ‡ ‡ ‡, very strong; ‡ ‡ ‡, strong; ‡ ‡, moderate; ‡, weak; , none. The S15-induced enhanced accessibility to RNase V1 at position 661-662 is indicated in the last column (V1). * and ** denote the observed reduction of reactivity of the naked RNA to hydroxyl radicals at positions 742-749 and 742-752, respectively. The different values for the wild-type RNA are given as references.

replaced by U. As expected, no footprint of S15 was observed at site 1 in any of these three RNA mutants (see Figures 6 and 7 for RNA G654C). However, no detectable footprint was observed at site 2, although this second site was unaffected by the mutations, indicating either that site 2 itself is not suf®cient to provide stable binding, or that an incorrect conformation of the three-way junction sterically impedes binding at this site. A less dramatic but still signi®cant effect was observed with the G752A substitution, which increased the Kd by 50-fold (Table 2). This might

be explained by maintaining the C754-G654 pair and one hydrogen bond between N3 of A752 with C754(N4). Surprisingly, the G752A substitution allowed partial restoration of a low level of binding to RNAs in which G654 was replaced by U or C. On the other hand, substitution of C754 by G could not be compensated by replacing G654 by C. Other attempts to substitute the original C-G
G base triple (by G
G-C or U
G
U) failed. Although all combinations were not tested, it appeared that the base triple is extremely constrained. The only RNA variants that retained

794

The rRNA Binding Site of Ribosomal Protein S15 Table 3. Effect of mutations on the af®nity of the rRNA for EcS15 (site 2 and other parts)

For details, see Table 2.

some binding (reduced about 50-fold) contained adenine at position 752. One might suppose that these combinations (C-G
A, C
C
A, C
U
A) allow unstable base triples or/and permit suf®cient ¯exibility that tolerate weak binding. On the contrary, other mutations that triggered stable incorrect conformations should not be recognized by S15. The unpaired G587 This nucleotide, which is conserved in 96 % of prokaryotic sequences, was initially assumed to be paired with the conserved C754. According to the crystal structure, the left unpaired G interacts with the phosphate group of C754. Here, we found that any substitution of this nucleotide did not affect signi®cantly the binding af®nity for S15 (Table 2). Consistently, the G587C substitution did not compensate for the C754G mutation. This result agrees with the fact that this nucleotide did not contact TthS15 in the crystal structure and that this base does not participate in building the bindingcompetent conformation. The bulged U653 As expected from its bulged out conformation, U653 was highly reactive to CMCT (Table 1). Consistently with the fact that this unconserved base does not interact with S15, its replacement by C had no effect on RNA conformation or S15 binding (Tables 1 and 2). However, deletion of U653 led to

a 19-fold decrease in binding af®nity, suggesting that this nucleotide plays an indirect role in S15 binding. Most likely, it acts as a spacer, allowing proper orientation of G654 for pairing with C754 (Nikulin et al., 2000). Unexpectedly, the deletion caused the appearance of strong reverse transcriptase pauses around positions surrounding the deleted position (C651, U652 and G654), preventing a correct interpretation of reactivity data at these positions (Table 1). Otherwise, no noticeable reactivity change was observed, with the exception of a slightly enhanced reactivity of A655 at both N1 and N7 positions, and a reduced accessibility to RNase V1 in helices H21 and H22 (Figure 7). In agreement with its altered binding af®nity, the hydroxyl radical footprint in site 1 was considerably reduced (Figure 6). This clearly indicates that the conformation of the junction has been altered in a way that allowed incomplete binding. Most surprising is the loss of detectable footprint at site 2 (Figure 6). One possible explanation is that the imperfect junction did not allow correct relative positioning of the two sites. Interestingly, no enhancement of RNase V1 cleavages was visible in this case (Figure 7), suggesting that the enhancement resulted from a local rearrangement requiring binding at both sites. Strikingly, this bulged residue appeared to be a very sensitive sensor of the three-way junction conformation, since its reactivity was altered in most of the RNA variants from site 1 that were affected in their binding properties (Table 1).

The rRNA Binding Site of Ribosomal Protein S15

795

Figure 7. Results of RNase V1 hydrolysis of different RNA fragments in the presence (‡) and in the absence ( ) of EcS15. The right lane is in the absence of RNase V1. The S15-induced protections are indicated by arrowheads, ®lled for strong protection, gray for moderate and empty for weak. Enhanced accessibility is denoted by (‡). Sites 1 and 2, as de®ned by hydroxyl radical footprints, are indicated.

The conserved reverse Hoogsteen U652-A753 pair The formation of a reverse Hoogsteen pair is fully supported by the high degree of reactivity of A753(N1), and the non-reactivity of U652(O2) and A753(N7) (Table 1). Mutations of this pair were found to impair S15 binding to different degrees (Table 1). The most severe effects were observed with the three A753 substitutions, in the order C > G > U (Table 2), causing a drop of af®nity from 600 (according to competition experiments) to 30-fold, respectively. All these mutations led to a destabilization of the reverse Hoogsteen pair and probably induced rearrangement of the junction, as denoted by the strong decrease of reactivity of U653 observed in RNAs A753C and A753U (Table 1). In the case of the A753C mutant, strong perturbations of its reactivity pro®le were observed. Pauses appeared at and near the mutated position, with a very strong one at the adjacent C754. On the other strand, beside the decreased reactivity of U653, that of G654(N1) and A655(N7) was slightly increased. The most unexpected effect concerned the hydroxyl radical reactivity pattern, which showed an almost complete extinction of reactivity between nucleotides 742

and 752 (Figure 6). In parallel, the accessibility of helices H21 and H22 to RNase V1 was decreased (Figure 7). Most likely, this behavior re¯ects an important rearrangement of the junction, which is not recognized by S15. Accordingly, no hydroxyl radical footprint (Figure 6) and no RNase V1 enhancement (Figure 7) were observed. The A753U mutant, which showed a 30-fold reduction of binding, also displayed a perturbed hydroxyl radical reactivity pattern, with a strong reduction between nucleotides 724-749 and 655-658. Nevertheless, a weak footprint was observed in site 1 (restricted to weak protections at positions 749-752), but not in site 2. Again, no RNase V1 increase occurred (Figure 7). The double mutation U652A-A753U allowed improved binding as compared to the single A753U mutation (from a 30-fold to a ®vefold reduction) (Table 2), although this RNA displayed the same perturbed hydroxyl radical reactivity pattern as the single mutant. It was not clear whether an inverted reverse Hoogsteen or a Watson-Crick pair could be formed in the naked RNA, since U753 and A652 showed a medium degree of reactivity at N2 and N7, respectively (Table 1). Again,

796

The rRNA Binding Site of Ribosomal Protein S15

Figure 8. Interaction between EcS15 and the mutant rRNA fragments. (a) Saturation curves with 32P-labeled wildtype RNA (*), mutant U740C (~), C739A-U740C (^), C754A (!) and 5 S rRNA from T. thermophilus (&). Binding curves for the two last RNAs are not shown, since saturation of binding was not reached during experiments and data could not be ®tted to the theoretical equation. (b) Competition between 32P-labeled wild-type RNA and unlabeled wild-type RNA (*), G656C-U657A-A749U-C750G (~), A753U ( & ), CP12 (!).

the reactivity of U653 was decreased, as in all the tested RNAs mutated in the junction. Therefore, it appeared that the inversion, although not restoring a wild-type conformation, might induce a conformation ¯exible enough to permit binding. The footprint in site 1 was reduced, and no footprint was detected in site 2. The single U652A mutant did not show perturbations of the hydroxyl radical reactivity pattern, and was able to bind S15 with a ®vefold reduced af®nity (Table 2). Its footprint in site 1 was reduced on the 30 -strand (no protection at positions 753-755), but more extended on the 50 strand, as denoted by increased protection at A655 and new protection at the adjacent G654. These subtle variations in the protection pro®le probably re¯ect a discrete rearrangement of the junction. In contrast to the other mutants, binding to site 2 was allowed with a reduced ef®ciency, accompanied by a weak RNase V1 increase (Figure 7). The conserved A655-U751 Watson-Crick base-pair The conserved base-pair A665-U751, although well de®ned in the crystal structure of the complex, displayed weak reactivity to chemicals in the free RNA (Table 1), probably re¯ecting some local instability in the proximity of the base triple in the absence of the bound protein. According to the crystal structure in which Thr24 (conserved in E. coli) hydrogen bonds with the 20 -OH group of U751, this pair is stabilized by the bound protein. The replacement of the A-U by G
U (A655G), or G-C (A655G-U751C) base-pairs, the two pairs most frequently found in natural RNAs after A-U, reduced the af®nity by a factor of 6 to 7 (Table 2). The formation of a wobble G-U or a G-C pair in these two RNA variants was supported by the non-reactivity of G655 at N1 (Table 1). However, minor perturbations of the reactivity pro®le were observed, in particular, a reduction of the reactivity

of the bulged U653 (Table 1), which revealed some change in the geometry of the junction. Strikingly, the (A655G-U751C) mutant also showed a reduced reactivity to hydroxyl radical cleavage (Figure 6), as in the case of (A753U) and (U652A-A753G) mutants. The sixfold reduction of af®nity was re¯ected by slight reduction in the hydroxyl footprint in site 1 (Figure 6). As already observed, small reduction of binding in site 1 was accompanied by weakening of the footprint in site 2 (Figure 6). Concomitantly, the enhancement of RNase V1 cleavage between the two sites was either lost (A655G,U751C) (Figure 7) or weakened (A655G). Meanwhile, destabilization of pairing by introducing the U751C mutation or attempts to form non-canonical purine-purine base-pairs (U751G, A655G-U751G) had a more dramatic effect and increased the Kd by 100-fold. Probing analysis of the U751C variant (Table 1) suggested an important rearrangement, compatible with the formation of a Watson-Crick C751-G654 pair (both unreactive), leaving A655 unpaired (increased reactivity at N1), and the formation of a wobble U653-G752 pair (both unreactive). Such a rearrangement of the bottom part of helix H22 would impair the formation of the base triple. The changes were accompanied by the same reduction of reactivity to hydroxyl radical cleavage as in the A655G-U751C mutant. The G656-C750/U657-A749 base-pairs These two canonical base-pairs are less conserved than nucleotides involved in the junction. G656-C750 match about 85 % and U657-A749 79 % prokaryotic 16 S rRNAs sequences. Together, they form a motif found in 68 % of the sequences. In T. thermophilus, they are replaced by C-G and G-C, respectively. As shown above, a very similar array of hydrogen bonds could be formed in the E. coli

797

The rRNA Binding Site of Ribosomal Protein S15

complex, although the interacting amino acid residues are unchanged in EcS15 (Figure 4). Here, we inverted the wild-type G-C/U-A base-pairs, a combination that is not found in nature. These mutations did not alter the reactivity pattern of the RNA, with the exception of a slight reduction of the reactivity of A747 (Table 1). The only remarkable change is a new RNase V1 cut at position 656 (Figure 7). The inversion of the G656-C750 basepair was expected to re-establish the T. thermophilus situation, whereas inversion of the U657-A749 pair did not. Indeed, the absence of an amino group at N2 in the mutated position 657 should prevent hydrogen bonding with Thr21 (Figure 4(b)). The footprint at site 1 was perturbed only slightly (Figures 6 and 7), in agreement with the moderate effect on binding (®vefold reduction of af®nity, Table 2). The signature of second-site binding (footprints and enhanced RNase V1 cleavage) was still observed (Figures 6 and 7), indicating that the relative orientation of the two sites is maintained. Site 2 does not significantly contribute to the stability of the complex The G-U/G-C motif None of the mutations introduced into the G-U/ G-C motif displayed as dramatic a defect as those observed in three-way junction (Table 3). The most severe effects (around 10 to 20-fold) were observed by mutations that introduced a mismatch at the G-C pair (G667C-C739A, G667C-C739U, C739A, C739A-U740C), probably due to a destabilization of adjacent G-U and of site 2 as a whole. On the other hand, inversion of G-C (G667C-C739G) or replacement of G-U by G-C (U740C) or G
G (U740G) all induced a modest reduction of binding (three- to ®vefold), suggesting that these mutations allow formation of compensating contacts. Interestingly, the inversion of the G-C pair (G667C-C739G) or the replacement of G-U by G-C (U740C), con®rmed by probing analyses, both induced a destabilization of the adjacent purineloop, re¯ected by the increased reactivity of AGA665 (Table 1). In the case of the G-C inversion, an increased accessibility to RNase V1 was observed in the vicinity of the mutation (Figure 7). Consistent with the moderate effect on binding, footprint at site 1 was unchanged. The existence of compensating contacts in site 2 was con®rmed by protection to hydroxyl radicals (Figure 6) and RNase V1 (Figure 7). However, the loss of the S15induced enhancement of RNase V1 cleavages (Figure 7 and Table 3) suggested that conformation changes promoted by S15 require correct binding at site 2 and/or a proper conformation of the purine loop. Mutations at both pairs (C739U-U740CC739A-U740C) had additive effects (Table 3). The contact between His45 and the 20 OH group of the adjacent nucleotide 668 was believed to be sequence-independent, since a C668-G738 pair was

substituting for the natural G-C pair in the short rRNA fragment. As expected, the inversion of the G668-C738 pair did not affect on EcS15 binding (Table 3). The conserved purine-rich interior loop Although this ®ve nucleotide interior loop is highly conserved, it provided only one contact with S15 in the T. thermophilus complex (Arg34 with the phosphate group of G742). However, this loop appeared to be crucial for subsequent protein binding during 30 S assembly (Nikulin et al., 2000; Agalarov et al., 2000). Indeed, the crystal structure of the extended complex, containing S6, S15 and S18, revealed a tertiary fold constrained by A665, which was ¯ipped out of helix H22 and inserted into helix H23a, forming a base-pair with G724 and being stacked on G725 (Agalarov et al., 2000). This fold was further stabilized by additional contacts with S15, between His50 and the GAAG tetraloop of helix H23a, and stacking of Arg53 below the purine ring of A728 (Agalarov et al., 2000). Interestingly, A665 was also bulging out in the crystal structure of the small S15 complex, despite the absence of helices H23a and H23b (Nikulin et al., 2000). This residue stacked on the homologous nucleotide of a symmetry-related molecule, while G664 was alternatively bulged out or making a non-canonical base-pair with G741. Thus, the bulged out conformation of A665 stabilized by crystal packing might reveal the ability of S15 to promote, or stabilize, the conformation of the purine loop with a ¯ipped-out A665. It is noteworthy that probing data on the unbound RNA did not favor a unique conformation with a ¯ipped-out A665 (Table 1). Nucleotides forming the interior loop were either unreactive or poorly reactive to chemicals, and both A665 and A663 displayed a moderate reactivity. A S15-induced conformational adjustment was further supported by the reactivity increase of A665 observed in the presence of S15 in the E. coli complex (Mougel et al., 1988). Consistent with the fact that the abovementioned additional contacts are not required for optimal S15 binding (Batey & Williamson, 1996a; Serganov et al., 1996), none of the mutations made in this area signi®cantly impaired S15 binding (Table 3), despite the dramatic alterations of its conformation revealed by chemical probing (Table 1). Those alterations included complete destabilization of the loop and the adjacent U662-A743 (G664
), imposing G664 in a bulged out conformation (A663C-A665C and AGG5C), or substituting the complete loop by two canonical C-G pairs (A663C-A665C-G664
). Consequently, the accessibility to RNase V1 was reduced or lost in the adjacent part of helix H23 and S15 binding failed to increase it (see Figure 7, RNAs A663CA665C and A663C-A665C-G664
). These alterations of the purine loop resulted in a reduced hydroxyl footprint at site 2 (Figure 6, Table 3). Thus, a proper conformation of the purine loop,

798 although not directly involved in S15 binding, is probably required to allow a correct binding at site 2, by providing an appropriate ¯exibility between the two sites. Moreover, it is essential by mediating the S15-induced conformational change required for S6 and S18 binding. The bulged A746 A conserved feature is a single unpaired nucleotide on the 30 side of the bottom part of the helix (Gutell et al., 1994), whose exact position or nature is not constrained. This bulged nucleotide is not directly recognized by S15. In T. thermophilus, C748 was found bulging out, while in E. coli either A746 or A747 could be unpaired. Both nucleotides were reactive to chemicals, with a higher degree for A746 (Table 1), suggesting a conformational heterogeneity in the free RNA. In agreement with phylogenetic observations, substituting U for A746 affected neither the reactivity pattern (Table 1) nor S15 binding properties (Table 3, Figures 6 and 7). Deletion of A746 reduced S15 binding by only twofold (Table 3). The deletion did not affect the reactivity pattern, but induced pauses of reverse transcriptase around the deletion (Table 1). The hydroxyl radical footprint indicated a standard binding at site 1 but no detectable binding at site 2 (Table 3, Figure 6). Instead, unusual enhanced hydroxyl radical cleavages occurred at surrounding positions 743-744 (Figure 6), as well as very strong RNase V1 cuts at positions 661-663, differing from those usually observed at positions 662-663 (Figure 7). The low effect of the deletion on binding af®nity is rather intriguing, considering the complete lack of binding at site 2. However, a more pronounced effect was reported in the case of the B. stearothermophilus system, in which the deletion of the bulged nucleotide decreased binding by 30-fold (Batey & Williamson, 1996b). Although we have no clear explanation for these discrepancies, this bulged residue, which is not recognized by S15, most likely plays an indirect role, as already suggested (Batey & Williamson, 1996b), i.e. as a spacer, providing some ¯exibility that facilitates mutual adaptation of sites 1 and 2.

Discussion The present work is a thorough investigation of the E. coli S15 rRNA binding site by directed mutagenesis, RNA probing and protein footprinting. A ®rst observation that emerged from this study con®rmed previous observations that S15 binding triggers important RNA conformational changes (Mougel et al., 1988; Serganov et al., 1996). These local and distal rearrangements were clearly described in the crystallographic structures of the T. thermophilus S15-rRNA complex alone (Nikulin et al., 2000) and in the presence of S6 and S18 (Agalarov et al., 2000). Our footprinting exper-

The rRNA Binding Site of Ribosomal Protein S15

iments revealed the signature of the upper threeway junction rearrangement and the presence of the helix H23a contacts. Thus, the conformation of the S15-rRNA complex is the same in the presence and in the absence of S6 and S18, stressing the pivotal role of S15 in the 30 S assembly. This study was aimed to investigate the role of conserved nucleotides in S15 recognition and to evaluate the relative contribution of the two sites. The results are summarized in Figure 9. It clearly appeared that site 1 brought the most important contribution to the binding free energy. This site Ê 2 accessible Ê 2 of protein (over 6040 A buries 1500 A surface) from solvent accessibility, as calculated by CNS (BruÈnger, 1998). Our results showed that any mutation of the invariant nucleotides forming the C754-G654
G752 base triple abolished EcS15 binding, or in the best cases reduced the af®nity by 50fold (CGA, CCA, CUA combinations), thus pointing to the base triple as the most crucial element for protein binding. Its prominent contribution resides in its central role in governing the threeway junction folding. The particular spatial localization of phosphate groups in the resulting bend of the backbone around C754 is precisely recognized by residues in helix a3 (Figures 7 and 9). This underscores the shape complementarity between RNA and protein as an important determinant of speci®city. Important base triples were observed in other RNA-protein and RNA-peptide complexes, such as HIV-1 TAR with arginine (Puglisi et al., 1992) or a Tat peptide (Long & Crothers, 1999), BIV TAR with a Tat peptide, HTLV Rex peptide with a RNA aptamer (Jiang et al., 1999), L11-23 S rRNA site (Conn et al., 1999; Wimberley et al., 1999), where they play a central role in building protein-binding sites. Shape complementarity was pointed out in several RNA protein complexes such as the L11-23 S rRNA (Conn et al., 1999; Wimberley et al., 1999), and the L30-mRNA complex (Mao et al., 1999) in which a sharp bend of the backbone is contacted by the protein. Interestingly, the left unpaired G587 can be replaced by any nucleotide without affecting binding. This agrees with the fact that this nucleotide is not contacted by S15, despite its high degree of phylogenetic conservation, suggesting a role in some other crucial function of the ribosome, distinct from S15 binding. The pivotal role of C754 in the geometry of the junction might be related to a potential switch of Watson-Crick pairing of C754 from G654 to G587, as suggested by Nikulin et al. (2000). Such an alternative conformation, which remains to be investigated, might be involved in ribosome dynamics or in preventing misfolding of RNA. In addition to the base triple, the conserved canonical A655-U751 and non-canonical U652
A753 pairs, contribute to the edi®cation of the junction, in particular by allowing stacking between G588, A655, G654 and A753. Thus, any mutation causing even small perturbations of stacking or/and modi®cation of the hydrogen bonding array had more or less severe conse-

The rRNA Binding Site of Ribosomal Protein S15

799

Figure 9. A summary of the present mutagenesis study. (a) The effect of mutagenesis on EcS15 binding is indicated. The free energy of binding is essentially driven by site 1, and site 2 is dependent on proper binding at site 2. Binding at both sites triggers a conformational adjustment in the purine-rich loop and in the upper three-way junction (not shown), creating further binding sites for proteins S6 and S18. (b) The proposed interactions in E. coli rRNA: red and blue arrowheads indicate contacts with phosphate groups and 20 OH ribose, respectively, and boxed bases indicate interactions with functional groups of bases. Arrowheads in pale red indicate potential contacts between phosphate groups and amino acids in the variable helix a1 that exist in the T. thermophilus complex. Compensatory contacts may be possible, as suggested by protection from hydroxyl radicals. Conservation of nucleotides is indicated as in Figure 1(a).

quences. Otherwise, the variable bulged U653, which is not directly recognized by S15, was thought to act as a spacer that facilitates the correct folding of the three-way junction. Other contacts in helix H22 involve recognition of G656-C750/U657-A749 base-pairs through the shallow groove by conserved residues of loop a1. The variability of the concerned sequence and the modest effect of nucleotide inversion underscore the adaptability of contacts between base functional groups and amino acid side-chains and illustrate how base substitution can create a different, compensating set of interactions. Another interesting example concerns the U1A and U2 snRNPs in which a conserved arginine residue interacts with different bases in the two complexes (Oubridge et al., 1994; Price et al., 1998). These kinds of adaptations render dif®cult the interpretation of mutagenesis studies in deducing information about RNA-protein interactions and their exact contribution to the binding energy (Draper, 1999). Taken together, our results allow mutations of nucleotides governing the junction to be sorted into three classes, depending on the extent of binding defects. The ®rst class of mutations caused relatively weak defects (5 to 20-fold reduction of binding). One can assume that they generated a locally disordered structure, ¯exible enough to permit slightly reduced binding. Mutations of the second class, induced intermediate defects (50 to 100-fold reduction of binding). Most likely, they should lead to a reorganization of the junction into a stable structure that differed from that of the

wild-type. However, residual interactions were still possible (for instance, with the shallow groove of helix H22 above the junction), provided that binding of the protein was not sterically impeded. Mutations of the third class caused a loss of detectable binding. They were assumed to generate incompetent structures, due to the loss of shape complementarity and the occurrence of steric clashes with the protein. An important ®nding is that the high phylogenetic pressure observed in the RNA three-way junction is due to severe structural constraints to maintain a precise and unique three-dimensional backbone folding and helix stacking required for tertiary structure complementarity between the two ligands. Surprisingly, constraints on the protein side appear to be less pronounced, since alternative sequences are able to accommodate the tertiary folding (Figure 3). This is consistent with the fact that the ribosome is a ribozyme (Nissen et al., 2000) and the view that protein S15 appeared later in the evolution, once RNA had evolved to provide an optimal conformation. Similar conclusions were suggested in the case of L25-5 S rRNA (Lu & Steitz, 2000). Site 2 contributes only modestly to the free energy of binding, since perturbation or absence of binding at this site only caused a threefold to 18fold reduction of af®nity. Site 2 is more restricted Ê 2 from the solvent. than site 1, shielding 1100 A Contrary to site 1, recognition is essentially sequence-speci®c. Both amino acid and nucleotide residues involved in the interactions are highly

800 conserved (Figures 2 and 9). The different contribution of the two sites can be explained by the lower number of contacts involved in site 2 than in site 1 recognition, and by the fact that contacts in site 2 are mainly hydrogen bonds, which are known to have a rather poor contribution to the free energy of binding (4.1 kcal mol 1). On the other hand, site 1 recognition involves several ionic interactions that provide a large contribution to the stability (Figure 9). Furthermore, binding to site 2 is dependent on binding to site 1, since mutations that prevented or strongly reduced binding at site 1 also precluded binding at site 2, as seen from footprinting experiments (Table 2). Thus, site 2 by itself does not appear to be able to ensure stable binding. Interestingly, the bulged A746 essentially serves as a spacer, since its deletion prevents binding at site 2, although not affecting binding at site 1. Otherwise, this bulged nucleotide is not conserved and even its location in helix 22 is variable. Thus, it is most likely necessary to provide some ¯exibility between the two sites, allowing proper relative orientation. The conserved purine-rich loop is not an element of recognition for S15, despite its high degree of conservation. It becomes evident, from the S15induced conformational changes observed in this region and from the crystallographic structure, that it is involved mainly in subsequent binding of proteins S6 and S18 during assembly of the 30 S subunit (Agalarov et al., 2000). Interestingly, the increase of RNase V1 cleavages in the purine loop, which can be considered as the signature of the conformational change, is lost or decreased when binding at site 2 is affected or when the conformation of the purine loop is altered. Thus, we assume that the role of site 1 is to anchor S15 to the rRNA, while the role of site 2 is to trigger a precise conformational adjustment of this loop, allowing tertiary interactions to take place (in particular, the A665-G724 pair). The presence of separate RNA sites with distinct roles is already well documented in the case of aminoacyl-tRNA synthetases, which evolved through the progressive addition of modules (Delarue & Moras, 1993). Interestingly, a repeated helix-turn-helix motif appended to eucaryotic aminoacyl-tRNA synthetases was reported to share structural homology with the central domain of protein S15 (Cahuzac et al., 2000). This extra domain, which displays RNA-binding properties, is believed to serve as a cis-acting tRNA binding co-factor. Ribosomal EcS15 also binds to its own mRNA, stabilizing a pseudoknot structure that exists in equilibrium with a stem-loop structure (Philippe et al., 1990). The only obvious analogy with rRNA found in the mRNA is a G-U/G-C motif, which has been clearly identi®ed as a speci®c determinant of S15 binding (BeÂnard et al., 1998). However, as the G-U/G-C motif is not suf®cient to provide S15 binding in rRNA, since the disruption of the pseudoknot, while keeping the G-U/G-C motif, abolishes binding and control (Philippe et al.,

The rRNA Binding Site of Ribosomal Protein S15

1995). Thus, EcS15 apparently recognizes two distinct sites on its mRNA, the G-U/G-C motif and a particular fold of the backbone provided by the pseudoknot structure. Although EcS15 is reasonably assumed to recognize the same G-U/G-C motif in both RNAs, the question of whether the pseudoknot geometry partly shares some features with the three-way junction remains to be investigated. The fact that S15 also recognizes a stem-loop structure in 23 S rRNA (Culver et al., 1999) underscores the multifunctional role of this protein and its versatility in recognizing different RNA targets.

Material and Methods Chemicals and enzymes CMCT and H2O2 were from Merck, DMS was from Fluka, DEPC was from Sigma, kethoxal and phage T4 polynucleotide kinase were from U.S. Biochemical Co. Acrylamide and N,N0 -methylene bis-acrylamide were from BDH Chemicals. Radiochemicals were from New England Nuclear. Phage T4 DNA ligase and restriction enzymes were from New England Biolabs. RNase V1 was from Pharmacia Biotech. Phage T7 RNA polymerase was puri®ed from the overproducing strain BL21/ pAR1219, following the protocol provided by F.W. Studier. AMV reverse transcriptase was from Life Sciences Inc. Thermostable DNA polymerase ``Goldstar'' was purchased from Eurogenetec. Construction of mutant DNAs Wild-type RNA fragment used in the study was obtained by in vitro transcription of a plasmid DNA containing nucleotides 584-757 of E. coli 16 S rRNA and ten nucleotides of extra sequence (CAGACGGAUC) at its 30 end under the control of phage T7 RNA polymerase T7 promoter (Mougel et al., 1988). Mutations were introduced either by site-directed mutagenesis (Kunkel, 1985) of the plasmid carrying the E. coli 16 S rRNA gene fragment or by PCR, using wild-type or mutant DNAs as templates, and 50 primer containing the T7 promoter and a 30 primer carrying the mutation of interest. PCR fragments were puri®ed by ion-exchange chromatography on a Mono-Q column (Pharmacia Biotech). Plasmid DNAs were cleaved by BamHI prior to transcription. All DNA mutants were sequenced. Preparation of biological material Uniformly labeled RNAs were prepared from PCR fragments or linearized DNA by in vitro transcription with T7 RNA polymerase in the presence of [a-32P] ATP (3000 Ci/mmol). After transcription, the reaction mixtures were treated by DNase I and RNAs were puri®ed by electrophoresis on 8 M urea/8 % polyacrylamide gel. Unlabeled RNAs were also obtained by in vitro transcription and puri®ed either by electrophoresis on 8 M urea/ 8 % polyacrylamide gel or by gel-®ltration on a Bio-Sil column (Bio-Rad). RNAs were renaturated at 42  C for 15 minutes in buffer A (50 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate, 270 mM KCl, 5 mM dithiothreitol, 0.02 % (w/v) bovine serum albumin) and chilled on ice. EcS15 was overproduced in E. coli BL21 (DE3) strain, from the pET11c vector (Studier et al., 1990) carry-

801

The rRNA Binding Site of Ribosomal Protein S15 ing the rpso gene under the control of phage T7 RNA polymerase promoter. The protein was puri®ed in one step using ion-exchange chromatography on CM-Sepharose, essentially as described for its thermophilic analogue (Serganov et al., 1997).

Protein binding assays Filter-binding assays were conducted in buffer A as described (Serganov et al., 1996), using 10,000 cpm of labeled RNA (<1 pM) and increasing amounts of EcS15. Retention of RNAs was generally 30-55 %. Non-speci®c retention of RNAs (1-3 %) was measured by ®ltering the complete reaction mixture in the absence of S15. The apparent dissociation constants (Kd) were determined as the concentration of S15 necessary to obtain half-saturation, assuming that complex formation obeys a simple bimolecular equilibrium. Results were ®tted with the equation:  ˆ ‰S15Š=…Kd ‡ ‰S15Š† where  represents the fraction of radiolabeled RNA bound to the ®lter. Competition experiments were performed by ®lter-binding assay with a constant concentration of S15 (6 nM), negligible concentration of labeled wild-type 16 S rRNA fragment (0.005 pM) and a variable concentration of unlabeled RNA (0.1 nM to 0.1 mM). Data were ®tted using equation (5) of Lin & Riggs (1972). Dissociation constants are the average of at least two independent experiments with a standard error of 30 %. Fitting of the experimental data to the theoretical equation was performed by Sigma Plot software (Jandel Scienti®c, San Rafael, USA). Radioactivity was quanti®ed using a BAS 2000 BioImager (Fuji).

Probing and footprinting RNAs were probed with RNase V1, DMS, CMCT, DEPC and hydroxyl radicals generated by Fe(II)-EDTA. A standard assay contained 10 pM RNA, and RNase digestion or chemical modi®cations were according to Serganov et al. (1996). In footprinting experiments with hydroxyl radicals and RNase V1, the S15-RNA complex was formed in the presence of sixfold excess of protein over RNA for one hour on ice. After reaction, RNA was extracted by phenol/chloroform and chloroform, and precipitated by ethanol at 20  C. Modi®ed sites or cuts were detected by extension with AMV reverse transcriptase of (50 -32P)-labeled primer hybridized to the unpaired 30 -terminal sequence or nucleotides 681-694 of the RNA fragment. Elongation controls were run in parallel in order to detect spontaneous hydrolysis and stops of reverse transcription. Resulting cDNAs were precipitated by ethanol, washed by 70 % (v/v) ethanol, dissolved in formamide sample buffer, heated for one minute at 90  C and analyzed on 8 M urea/8 % polyacrylamide gels.

Modeling the E. coli complex The E. coli S15-rRNA complex was modeled from the T. thermophilus complex (Nikulin et al., 2000) (accession number 1DK1). Sequence changes in both protein and RNA were introduced by using program O (Jones et al., 1991). Figures were made by WebLab View Lite (MSI).

Acknowledgments P. Romby and E. Westhof are thanked for helpful discussions and advice. We are indebted to C. Cachia (Dijon, France) for providing S15 puri®ed from E. coli ribosomes. This work was supported by the Centre National de la Recherche Scienti®que (CNRS). A.S. was successively a fellow of the CNRS within the FrenshRussian Scienti®c Collaboration framework and from the Fondation pour la Recherche MeÂdicale (FRM).

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Edited by J. Karn (Received 21 September 2000; accepted 21 November 2000)