An Intricate RNA Structure with two tRNA-derived Motifs Directs Complex Formation between Yeast Aspartyl-tRNA Synthetase and its mRNA

doi:10.1016/j.jmb.2005.09.063

J. Mol. Biol. (2005) 354, 614–629

An Intricate RNA Structure with two tRNA-derived Motifs Directs Complex Formation between Yeast Aspartyl-tRNA Synthetase and its mRNA Michae¨l Ryckelynck, Benoit Masquida, Richard Giege´ and Magali Frugier* De´partement ‘Machineries Traductionnelles’, UPR 9002 Institut de Biologie Mole´culaire et Cellulaire du CNRS and Universite´ Louis Pasteur 15, rue Rene´ Descartes, F-67084 Strasbourg Cedex, France

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyltRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5 0 extremity of AspRS messenger RNA (mRNAAspRS) by its translation product and adaptation to the cellular tRNAAsp concentration. Here, we have established the folding of the w300 nucleotides long 5 0 end of mRNAAspRS and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNAAspRS folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNAAsp anticodon-like stem–loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNAAsp. Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: aspartyl-tRNA synthetase regulation; messenger RNA; tRNA mimicry; RNA structure; aspartate identity determinant

Introduction Protein synthesis involves numerous macromolecules, whose concentrations have to be properly adjusted with respect to cellular requirements and environmental variations. The coordination of their synthesis implies sophisticated regulation mechanisms to equilibrate the different partners. Amongst the mechanisms allowing such Abbreviations used: mRNAx, messenger RNA coding for protein x; aaRS, aminoacyl-tRNA synthetase; UTR, untranslated terminal region; ORF, open reading frame; CMCT, 1-cyclo-hexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate; DMS, dimethyl sulfate. E-mail address of the corresponding author: [email protected]

adaptations, feedback regulations are fast and economic solutions. Such mechanisms have been first demonstrated for prokaryotic RNA-binding ribosomal proteins. These proteins recognize their own mRNAs leading thereby to translational inhibitions by hindering ribosome fixation on the message.1 Well-known examples are the regulation of Thermus thermophilus and Escherichia coli protein S152,3 as well as archaeal L1 proteins4 and E. coli protein L4.5 This is also the case for several aminoacyl-tRNA synthetases (aaRSs),6 the key enzymes in protein synthesis, which ensure correct expression of the genetic code by specifically recognizing and charging their cognate tRNAs.7 Here, the seminal case is the regulation of E. coli ThrRS.8 All these feedback regulations require and have in common the presence of

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

615

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

specific RNA domains in their mRNA that are recognized by the encoded protein. These domains correspond, at least partially, to structural mimics of the cognate RNA substrate of the regulated proteins; namely, regions of 16 S or 23 S ribosomal RNAs for ribosomal proteins or cognate tRNA for aaRSs. In eukaryotes, feedback regulation is more elaborate, since mRNAs can be targeted in different sub-cellular compartments corresponding to the different stages of their functional lifecycle. Thus, protein binding to mRNA can inhibit transcription, maturation, export, degradation or translation of the mRNA in the nucleus or in the cytoplasm. For example, regulation of yeast L30 and L32 ribosomal proteins implies recognition of a specific RNA structure (23 S rRNA mimic), which modulates the nuclear splicing of their own mRNAs.9,10 Saccharomyces cerevisiae AspRS is the first example of a eukaryal aminoacyl-tRNA synthetase regulated via a feedback mechanism. This AspRS, not only binds specifically in vitro to the 5 0 end of its own mRNA but does it also in vivo.11 The consequence is a reduced cellular concentration of the synthetase. This regulation acts at the level of mRNAAspRS accumulation and specifically depends on the cellular concentration of tRNAAsp. Moreover, the nuclear localization of a fraction of AspRS led us to propose that AspRS inhibits the transcription of its coding mRNA.12 Interestingly, binding of the mRNA fragment with AspRS parallels what is already known about the recognition of tRNAAsp by AspRS. Indeed, both complexes involve a lysine-rich 70 amino acid residues domain located at the N terminus of the synthetase (Figure 1(a)). This extension is essential for the synthetase to bind the mRNA and increases 100-fold its affinity for tRNAAsp.11,13 Moreover, only tRNAAsp can dissociate the complex between AspRS and its mRNA. To explore the structural basis of the mRNAAspRS recognition by its encoded enzyme and to determine whether this RNA contains a mimic of tRNA, we have established a model of the secondary structure of the region in mRNAAspRS (mRNA(K38,C 250) ) that has been shown to interact with AspRS.11 This folding model relies mainly on structural probing of the RNA with structure-sensitive nucleases and base-specific reagents.14 Further, we have identified in this RNA the domains in close vicinity to the bound AspRS. We show that one of them contains a tRNAAsp-like fold in which four out of the six aspartate identity elements are conserved in an anticodon-like loop. Furthermore, and in agreement with a three-dimensional model of the mRNA domain, we suggest that the interaction implies a motif repeated twice in the RNA and spaced enough to be independently bound by each monomer of the enzyme. Finally, we show that the elements recognized by S. cerevisiae AspRS on its mRNAAspRS are conserved in different Saccharomyces species.

Results and Discussion Size of the regulatory mRNA fragment and its reduced solubility It has been shown that the minimal size of mRNAAspRS required for efficient AspRS binding encompasses the last 38 nt of the 5 0 UTR and the first 210 nt in the open reading frame (ORF) (Figure 1(a)).11 Delineation of this minimal domain is also supported by sequence conservations in mRNAAspRS of different Saccharomyces species (Figure 1(b)). Conservation is strong in the ORFs but diverges significantly in the 5 0 untranslated regions (UTRs). For technical reasons, structural probing of the regulatory region was done on a larger molecule covering nucleotides K38 to C330. In this RNA, nucleotides 210–330 are dispensable for efficient and specific formation of the AspRS/ mRNAAspRS complex. But this sequence stretch, even if not essential, stabilizes the complex to some extent, as seen in band-shift experiments where complex dissociation is more important with shorter RNAs.11 In other words, whether nucleotides 210–330 are present or absent does not affect the affinity in the complex, but their presence increases the quality of the band shift. Possibly, this sequence does not participate in binding but helps folding of the minimal RNA domain (K38,C 210). Moreover, this hypothesis finds support in the fact that mRNA(K38,C330,D4–210), containing only the 5 0 UTR and nucleotides 210–330, does not bind at all to AspRS (as seen at the top of Figure 4). With these considerations in mind and based on enzymatic (RNase V1 and T2) and chemical (Dimethyl sulfate (DMS) and 1-cyclo-hexyl-3(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT)) probing, we established the secondary structure of the wild-type molecule in the K38 to 250 region. Notice that this molecule is fully soluble only in phosphate buffer (25 mM at pH 7.5, the condition used for enzymatic probing and band shift assays) as seen by gel-filtration chromatography (data not shown). In Tris or borate buffers, required for chemical probing, its solubility is severely impaired. Despite these solubility variations, probing data obtained with nucleases and chemicals were consistent and allowed us to draw the secondary structure of the molecule. Secondary structure of mRNA(K38,C270) Figure 2(a) displays typical gels with RNase V1 cleavage patterns (this RNase probes specifically structured regions) that support the global folding of the molecule. Based on repeated structural probing with enzymatic and chemical probes, individual RNA subdomains were delimited on mRNA(K38,C330) (only the region K38 to C250 was carefully analyzed) (Figure 2(b)). Each helical element of the subdomains was confirmed by a systematic mutational study (data not shown). As a

616

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Figure 1. Structural background underlying the interaction between AspRS and its two substrates, mRNAAspRS and tRNAAsp. (a) The Figure highlights correlations between the mRNAAspRS sequence and the crystal structure of the translated AspRS subunit (in yellow) to which a flexible N-terminal domain is appended (in blue). Notice the presence of an RNA-binding motif (in magenta) in the central helix of this domain.11 (b) Sequence alignments of mRNAAspRS from five Saccharomyces species with their 5 0 UTRs indicated in green and the coding sequences in black. Numbering is according to the sequence of S. cerevisiae mRNAAspRS. The minimal sequence within mRNAAspRS recognized by the synthetase is delimited with red triangles. Conserved sequences involved in a long-range interaction between the 5 0 UTR and the coding sequence are boxed in red (for details see Figures 2(b) and 7; notice semi-conservation at position K28 allowing G–C or G–U pairings). Other conservations are indicated by a star (*). Sequences of mRNAs were retrieved from yeast genomes (http://cbi.labri.fr/Genolevures/).

typical example, mutations that disrupt potential Watson–Crick base-pairings within sequence 65UUGGG69 in stem P7, lead to complete disappearance of the RNase V1 cleavages in this stem. Likewise, mutations in sequence 199GCCG202 inhibit RNase V1 cleavages in 225CGGU228 and vice versa mutations in 225CGGU228 inhibit RNase V1 cleavages in 199GCCG202, strongly supporting existence of a pseudo-knot in subdomain IIb. There are mutations in J4-5 that destabilize the G–U rich part of P4 but not P5. Similar experiments support the existence of P9, P10, P16 and P17 (data not shown). Most often, the proposed stem–loop structures were consistent with structures predicted by the M-fold software.15 The folding emphasizes the binary organization of mRNA(K38,C250) with two major sectors (domain I,

encompassing nucleotides K10 to 134 and domain II divided into two parts, namely bipartite subdomain IIa from K38 to K24 and 150 to 196, and subdomain IIb from 197 to 250) linked together by two floppy connectors C1 and C2 (K26 to K11 and 135–150). These connectors are composed of purinerich stretches and are strongly reactive to chemicals (DMS alkylates N1 in adenine and N3 in cytosine, and CMCT, N3 in uridine and N1 in guanine) and to RNase T2 (cuts single-stranded regions), and not cleaved at all by RNase V1, highlighting their nonstructured nature. The peculiar base composition of the connectors and their floppy nature could account for the solubility problems met during this study. In domain I, the RNA (from K10 to 40, with P3, P4, P5 and the corresponding junctions) adopts a pseudo-knotted conformation, containing

Figure 2 (legend next page)

618

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Figure 2. Structure probing and folding model of S. cerevisiae mRNA(K38,C270). (a) Typical autoradiographs of RNase V1 cleavage patterns in different regions of mRNAAspRS. Probing experiments were performed with increasing RNase V1 concentrations. RNA sequencing as well as controls without RNases were run simultaneously. The structural organization of the RNA sequences is schematized at the right of the gels, with P meaning paired in helices, J junctions and L apical loops. (b) Folding of mRNAAspRS in the K38 to 250 region with all mRNA cleavages/modifications generated by RNase V1 (:), RNase T2 (C) and by CMCT/DMS (B) indicated on the sequence. The Figure shows the partition of the molecule into two domains, each of them being divided into subdomains. The cleavage patterns were conserved amongst repeated experiments (at least triplicates), but intensities of cuts could vary from one experiment to the other. Here, we have indicated averaged intensities of signals calculated from at least three independent probing experiments. They are categorized into three families: (i) yellow corresponds to cleavages appearing only in the presence of the highest RNase or chemical concentrations; (ii) red to cleavages with an intensity comparable to the strongest signals in RNA sequencing experiment; and (iii) orange corresponds to intensities in between. Notice that wild-type residue AK38 was changed to CK38 for cloning constraints. In both (a) and (b), the 5 0 UTR sequences are in green and the sequences making the long-range interaction in domain II are on a blue background.

the AUG initiation codon and defines subdomain Ia. The second half of domain I delineates subdomain Ib (from 37 to 134, with P6, P7, P8 and the corresponding junctions and loops) and folds in three stem–loops. As pointed out above, domain II is also divided into two parts, with subdomain IIb (nucleotides 204–270 with P15, P16 and P17) not required for synthetase binding. This region folds per se and can be removed from wild-type mRNA without affecting the binding to AspRS11 and damaging its overall structure. It is highly structured, since the majority of signals obtained were RNase V1 cleavages (Figure 2(a)). Subdomain IIa (K38 to K27 and 150–196) appears also well structured in an elongated hairpin. Interestingly, subdomain IIa is of chimerical nature and contains a helical domain (P1 and P2)

involving long-range interactions between residues K38 and K28 from the 5 0 UTR and nucleotides 182– 196 from the ORF. We are aware that the existence of helices P1 and P2 (highlighted in blue in Figure 2(b)), besides the potential Watson–Crick base-pairings, is supported by only a few RNase V1 signals. But the proposed structural arrangement answers questions raised by the functional analysis where deletion of the 5 0 UTR sequence inhibits AspRS binding completely (see later). Structurally, the strongest evidence supporting the existence of helices P1 and P2 comes from the comparison of cleavage profiles obtained with the full-length molecule and with a shorter molecule covering nucleotides 1–210 (mRNA(1,C330) was not soluble). The size of mRNA(1,C210), however, was not sufficient to observe the decisive disappearance of RNase V1 cleavages at residues 194–198. But the

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

619

Figure 3. AspRS footprint on mRNAAspRS. (a) and (b) Autoradiographs corresponding to the footprint of AspRS on C1 and domain Ia (nucleotides K38 to C41) (a) and C2 and domain IIa (nucleotides C135 to C192) (b). The modular mRNA architecture as defined in Figure 2 is schematized at the left of the gels. Asterisks (*) at the right of the gels indicate RNA degradations. (c) Model of secondary structure of mRNA, with protections against RNase V1 (:) and RNase T2 (C). Protection strengths are indicated by light blue (weak protections) or dark blue (strong protections) symbols. Intensifications of cuts are indicated by red symbols.

620

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Figure 4. Deletions in mRNAAspRS and their effects on binding full-length AspRS. In the right half, the 5 0 UTR sequence is drawn in green, whereas sequences mutated or deleted are indicated in red on the mRNA schemes. On the left side, the Kd values as well as the corresponding gel-shift experiments are shown. Notice that Kd values obtained for mRNA fragments already tested11 differ because of increased concentrations in salt and RNA competitors in the present work (see Materials and Methods).

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

621

existence of sequence conservation in the 5 0 UTR (Figure 1(b)) as well as the presence of enhanced signals in P11, P12 and P13, and the signal lost at G184 (data not shown) show clearly that the 5 0 UTR deletion changes strongly the accessibility of this stem–loop to RNases and points to an intricate structure in the presence of the 5 0 UTR.

that protections vary in intensity depending on their location in the messenger with strongest protections in subdomains Ia (P4, J4-5 and G28 in J3-5) and IIa (P2) while RNase T2 cleavages in the apical loop L4 are not affected. Finally, disappearance of RNAse T2 signals in C1 and C2, with sequences fully (AK27–AK24) or partly (AK23–AK17, A135–G149) protected, indicates that this region is also in close vicinity with the synthetase. The quasi-symmetric organization of the mRNA/ AspRS complex is noteworthy. This symmetry is corroborated by the increased accessibility, in the presence of AspRS, towards nucleases of critical positions in both RNA domains (nucleotides 40 and 41 in J5-6, 131 in P9, 191 and 192 in J2-1).

AspRS footprint on its mRNA As mentioned above, mRNA(K38,C330) is fully soluble only in phosphate buffer. Thus, footprint analyses were conducted with RNases, since phosphate-specific probes could not be used. Despite an intrinsic lack of precision of the bulky enzymatic probes and the impossibility of distinguishing direct interactions from steric hindrance effects preventing RNase action, experiments yielded clear protection patterns. Typical footprints seen in Figure 3(a) and (b) cover residues K38 to 196 (subdomain IIb was not probed because it does not participate in synthetase binding). They reveal protections in both helical and singlestranded regions of the RNA. Altogether, the results show that protections are concentrated in subdomains Ia and IIa, and the single-stranded connectors C1 and C2 (Figure 3(c)). Interestingly, subdomain Ib is not protected against RNases, indicating that it is not in close proximity with the synthetase in the mRNA/AspRS complex. Notice

AspRS recognizes two independent domains on mRNAAspRS The regions protected by AspRS were mutated extensively in mRNA(K38,C330) to determine more precisely the sequence stretches important for recognition. The direct effect of the mutations on AspRS binding was measured by electrophoretic gel mobility-shift assay (Figure 4). Whereas, the wild-type molecule, mRNA(K38,C330) is characterized by a Kd of 90 nM, weakening the pseudoknot architecture in subdomain Ia by replacing (K38,C330,mutant1) ) 10GACG13 by 10AAAA13 (mRNA increases the affinity slightly (K d of 60 nM).

Figure 5. Identification of the two binding regions in mRNAAspRS and their sequence homologies with tRNAAsp. The secondary structures of (a) mRNAdomain II, (b) mRNAdomain I and (c) tRNAAsp are compared. Sequence homologies between mRNAdomain I, mRNAdomain II and tRNAAsp are indicated in green and homologies between mRNAdomain I and tRNAAsp are indicated in red. The contact regions of the RNAs with AspRS as defined by protection patterns against RNase V1 cleavage determined in this study on the mRNA molecule or on tRNAAsp,13 are shadowed.

622

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Table 1. Binding of mRNAAspRS constructs on minimalist AspRS1–207 restricted to anticodon binding module prolonged by the N-terminal extension mRNAAspRS constructs RNA A. Full and fragmented mRNA Full mRNA Domain I Domain II B. Mutated domain I Mutant 1 Mutant 2 Mutant 3 Mutant 4

Kd

wt-sequence

Mutation

(nM)

(n-fold)

K38,C330 K38,C97 K38,C270

– – D4–250

90 370 250

1 4 3

10GACG13/10AAAA13

250 1300 2000 n.m.

3 14 22 [22

K38,C97 K38,C97 K38,C97 K38,C97

22GUC24/22UAU24 2UGUCU6/2ACAGA6

D2–25

Deletions in subdomains Ia or IIa did not affect the AspRS binding more than two- or threefold, as exemplified with mRNA(K38,330,D2–25) or mRNA(K38,330,D151–179). Only two variants affect AspRS binding significantly. They correspond to mRNAs deleted of the entire 5 0 UTR domain (mRNA(1–210)) or deprived of the heart of the structure with only the 5 0 UTR and nucleotides 210–330 preserved (mRNA(K38,C330,D4–210)). From what precedes it can be conjectured that subdomains Ia and IIa act independently towards the two AspRS subunits. The presence of the long floppy connectors between these two structures is in good agreement with this hypothesis. Moreover, this hypothesis agrees with the absence of significant losses in binding when each subdomain is mutated separately. Indeed, only removing the 5 0 UTR affects both of them by disrupting the pseudo-knot in subdomain Ia and removing P1 and P2 in subdomain IIa. To confirm this prediction, two half-mRNAs were produced (Figure 5). The first one is a construct described that contains only the 5 0 UTR region (nucleotides K38 to C1 and 150–270) 11 and designated here mRNAdomain II (Figure 5(a)), the second one being mRNAdomain I corresponding to the mRNA shortened at position 97 (nucleotides K38 to 97) (Figure 5(b)). Both mRNAdomain I and mRNAdomain II are poorly recognized by AspRS (data not shown), presumably due to steric hindrance between AspRS and mRNA regions improperly folded locally. However, as shown, a synthetase reduced to only its anticodon binding domain and the N-terminal extension (AspRS1–207) binds mRNA(K38,C330) with the same efficiency and specificity as full-length AspRS.11 This minimalist enzyme is monomeric, since the motif involved in dimerization, essentially present in the catalytic domain of AspRS,16 is lacking. It is shown here that AspRS1–207 binds efficiently and independently each half of the mRNA fragment, as reflected by the Kd values only three- to fourfold higher than for mRNA(K38,C330) (Table 1). On the other hand, the presence of structural similarities in subdomains Ia and IIa raises the possibility that both of them could be recognized by AspRS in a similar way. This view finds support in

the sequence conservation within stems P4 (182GUGA186 base-paired to K31UUGUK28) and P2 (K3GUGA1 base-paired to 20UUGU23) in subdomains Ia and IIa (highlighted in green in Figure 5). Both are surrounded by open structures (J4-5 and J2-1, respectively) and are the sites of strongest protection by AspRS. Moreover, competitions show that mRNAdomain I and mRNAdomain II compete efficiently with each other for AspRS binding (domain I displacing domain II from AspRS1–207 and reciprocally, with a Ki of 1000 nM only fourfold higher than that for mRNA(K38,C330)). Subdomain Ia in mRNAAspRS shares strongest homologies with tRNAAsp Structural similarities exist between the mRNAAspRS and tRNAAsp (Figure 5). The tRNAAsp regions strongly protected against RNase V1 cleavage in footprint experiments with AspRS are located in the anticodon stem and loop.13 Interestingly, if the sequence homologies are uncertain between the conserved stems of subdomains Ia and IIa and the anticodon stem of tRNAAsp, the resemblance is substantial between sequence 2UGUCUC7 in J4-5 of subdomain Ia and the tRNAAsp anticodon loop (33JGUCGC38) (highlighted in red in Figure 5). Even if of different size, both loops possess at equivalent positions 4 nt known to be part of the aspartate identity set;17,18 namely, the three anticodon bases 34GUC36 and C38 the last residue of the loop (homologous to AspRS , Table 2). Identity 3GUC5 and C7 in mRNA determinants in tRNA were found in contact with the synthetase in the crystal structure of the Table 2. Correspondence between nucleotides in yeast mRNAAspRS and the anticodon loop of cognate tRNAAsp tRNAAsp J32 U33 G34 U35 C36 M1G37 C38

mRNAdomain I

mRNAdomain II

A1 U2 G3 U4 C5 U6 C7

A185 A186 A187 A188 G189 A190 U191

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

623

AspRS/tRNA Asp complex.19 Moreover, the presence of U2 in the messenger, equivalent to strictly conserved U33 in tRNAs known to participate in the anticodon-loop organization,20,21 reinforces the notion of anticodon loop mimicry and suggests that domain Ia could be recognized by AspRS in the same manner as in the tRNAAsp anticodon domain. From another viewpoint, one notices that the sequence C1 to 20 can be compared to an intron as found at an equivalent position in many eukaryotic tRNAs.22 Interestingly, enzymatic probing of subdomain Ia suggests the existence of an equilibrium between two structures (“disrupted” and “closed” pseudo-knot) because of RNase V1 signals in J4-5 (Figure 2(b)). Disrupting P5 in the pseudo-knot allows Watson–Crick pairings between 2UGUC6 and 10GACG13, with the consequence of generating an enlarged helical stem with four additional basepairs. This idea is well supported by the observation that short yeast tRNA introns are usually able to form additional base-pairs at the end of the anticodon stem. In fact, the role of the closed pseudo-knot would be to prevent base-pairing between 2UGUC6 and 10GACG13. In the case of a disrupted pseudo-knot, the mimicry with a tRNA anticodon stem would be lost and AspRS would no longer be able to recognize the GUC identity determinants buried in the resulting doublestranded structure. This prediction finds support in the three-dimensional model of the RNA (see below). However, when the sequence 10GACG13 is replaced by 10AAAA13 (mutant 1), P5 can partially form, whereas its base-pairing with 2UGUC5 is not recoverable. Interestingly, this mutation improves slightly (Table 1), but systematically, the affinity of domain I for AspRS, suggesting that hindering internal pairings in J4-5 allows a better presentation of the identity determinants to the synthetase. Existence of this structural mimicry is further supported by the properties of mutants (Table 1) that destabilize stem P4 (mutant 2) or modify J4-5 (mutant 3). Both mutations decrease the affinity of AspRS1–207 for domain I. However, only combination of the two mutations (mutant 4 with D2–25) abolishes completely recognition by AspRS1–207, suggesting that both the stem and its apical loop are important for subdomain Ia recognition.

dimeric AspRS. Modeling also took into account the key role of the AspRS N-terminal domain, since its removal completely abolishes the recognition of the messenger by AspRS. Altogether, modeling relied on the correct identification of the tRNA-like features in the messenger. They were easily identified in domain I (see above) but appear more obscured in domain II. However, the fact that internal loop J2-1 bulges out from stem P2 in a way similar to J4-5 from P4, is a good indication that it could mimic the anticodon loop of tRNAAsp. Hence, the binding mode of the mRNA on AspRS resembles that observed in the case of E. coli ThrRS,24 in which two distinct regions of the mRNA also mimic a tRNA, each docking onto the anticodon binding site of the synthetase.

A model of the regulatory region in mRNAAspRS in interaction with AspRS The modeling process Modeling the regulatory region of mRNAAspRS was based on (i) probing data of the free and AspRS-bound RNA, (ii) availability of the crystallographic structure of yeast AspRS19,23 and (iii) the hypothesis that the recognition of the messenger by AspRS mimics that of tRNAAsp. This hypothesis finds strong support in the quasi-symmetric organization of the regulatory domain of mRNAAspRS that mimics the two tRNAAsp molecules binding to

Generating the model The overall model (Figure 6, top) was obtained by individual modeling of domains I and II as defined by footprinting and their connection when docked on AspRS. Domain I was generated step by step. First, the main stem from subdomain Ia (P3 and P4) was built as two regular helices connected by a short internal loop composed of a 2 nt bulge (AK5 and CK4) on the 5 0 strand (J3-4) and a 1 nt bulge (C24) on the 3 0 side (J4-3). A search in structural databases allowed us to pick up a region from loop 1470 in 23 S rRNA from Haloarcula marismortui25 displaying exactly the same sequence. Based on this similarity, a Watson–Crick cis C–C base-pair should form between CK4 and C24 (see Leontis et al.26 for base-pair nomenclature), where the N4 atom of CK4 H-bonds to the N3 atom of C24. Thus, the AK5 residue from the bulge stacks between base-pairs (UK6–A25 and CK4–C24), inducing a slight overtwist in the helix at this step. Nucleotides identified in domain I by RNase V1 footprints were used to dock the entire helix onto the enzyme (PDB ID 1ASY) so as to overlap with the corresponding residues of tRNAAsp in the crystal structure of the complex. For that, the residues from J4-5 were superimposed on those from the tRNA anticodon loop they mimic (Table 2). The next step used interactive molecular modeling to perform a conformational search of the pseudo-knot accounting for the closure of its second stem (P5). In the resulting conformation, P5 occupies a region corresponding to the tRNA D-loop and the single strand connecting P5–P3, and with P4 passes along the side of the deep groove of the stem formed by P3 and P4 (Figure 6). Since the three remaining hairpins of subdomain Ib do not apparently bind to the enzyme, and because of the lack of constraints to explore their relative positions, they were not included in the modeling. Domain II was modeled on the second monomer of the AspRS complex following a strategy similar to that employed for domain I. Helical P2, identical with P4 in domain I, was superimposed on the anticodon stem of tRNAAsp. Residues from J2-1 (A 186–G 189) were then modeled in the same

624

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Figure 6. Three-dimensional model of mRNAAspRS interacting with dimeric AspRS and mimicry with the interaction of tRNAAsp on AspRS. Top panel: Model of the complex. The backbone of the mRNA (with sketched bases) docked on the crystal structure of AspRS (as in the complex with tRNAAsp) is emphasized in green (5 0 UTR) and orange (ORF). Notice the structural differences of the two RNA domains interacting with AspRS and the participation of the helices from the enzyme N-extension in the binding (well visible in the case of domain II). Left panel: Superimposition of the model of mRNAAspRS subdomain Ia (in green and orange) with the crystal structure of tRNAAsp (in white). Perfect superimposition concerns both RNA backbone and nucleotides in the tRNA anticodon loop and their mimics in the messenger. Right panel: Close-up view of the mimic of the anticodon branch of tRNAAsp in mRNAdomain I in contact with helices from the N-terminal extension of AspRS and its OB-folded anticodon binding module. The Figure highlights the proximities of the RNA with AspRS as defined by protections in footprints (blue spheres and golden spheres indicate strong and weaker protections, respectively).

conformation than the homologous residues from the tRNAAsp anticodon loop (Table 2). Stem P1 could then be added together with residues A190 and A191 from J2-1 not yet incorporated into the model,

although P1 was constructed just to check whether or not it was causing steric clashes with the enzyme. In a last step, the two domains I and II were linked together. The connector C1 was incorporated

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

625

into the model so as to link the two domains together. Due to its length and the relative positions of the 5 0 and 3 0 ends of domains I and II, respectively, the path of C1 was chosen on the side of the catalytic domains of the AspRS dimer. This connector was modeled as an unstructured RNA stretch, but with a correct stereochemistry.

enlarging by forming additional base-pairs within J5-4 and thus, enables bases corresponding to tRNAAsp identity determinants to bind the enzyme. The N-terminal domain of AspRS is crucial for anchoring subdomain Ia on the protein. Domain II contacts the second subunit of AspRS by the sole P2 helix from subdomain IIa. Notice that bulge J2-1 connecting P2 to P1 is not protected at all by AspRS against nuclease cleavage. Instead, its accessibility to RNase T2 goes up, as shown by the intensification of the cleavages (Figure 3). In this model, one does not find any equivalent of a tRNA amino acid acceptor arm. This situation resembles that found in the ThrRS system,24,27 where the loss of the interactions between the acceptor arm of tRNAThr and ThrRS is compensated by a more intimate docking of the mRNA regions mimicking the

Key features of the 3D model of the regulatory region in mRNAAspRS The model in Figure 6 emphasizes the two stems constituting subdomain Ia. They are part of a pseudo-knot structure that has been footprinted by AspRS bearing the wild-type N-terminal domain. The pseudo-knot likely prevents P4 from

Figure 7. Sequence conservation in different yeast mRNAAspRS. The 5 0 extremity of mRNAAspRS from Saccharomyces (stricto sensus) species based on comparison of the sequences from S. cerevisiae, S. paradoxus, S. mikatae, S. bayanus. Sequences were folded and numbered according to the structural model determined for S. cerevisiae mRNAAspRS. The structure and sequence conservations in the RNA recognized by AspRS are highlighted with colors: green (conserved sequences in P4 and P2), red (anticodon-like sequence), blue (P5 in subdomain Ia and involved in the pseudo-knot) and blue background (the stem P1/P2 formed by the long-range interaction between 5 0 UTR and ORF sequences).

626

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

anticodon stem–loop onto the enzyme. Finally, one notices that symmetry is broken in the AspRS/ mRNAAspRS complex, since the equivalent anticodon-binding modules together with the protein N-terminal extension recognize two different RNA structures, although P2 and P4 share equivalent helical regions.

of the ThrRS/operator complex shows clearly that apart from the specific interactions with the anticodon-like loop, the ThrRS residues involved in the recognition of the rest of the mRNA structure are different from those involved in tRNAThr recognition.27 This is also the case for the E. coli S15 ribosomal protein. In this system, the region mimicking 16 S RNA is a G–U/G–C-like motif, while the second part of the binding-site is original and found only in the mRNA structure.28 In the present case, the mRNAAspRS of S. cerevisiae is recognized by AspRS through a set of signals displayed in two structurally independent domains spaced by two single-stranded connectors. A conserved double-stranded motif in subdomains Ia and IIa (P2 and P4) that is protected upon AspRS binding plays a key role in the interaction. Importantly, the symmetrical organization of the regulatory region in mRNAAspRS implies that each of these motives is recognized by one subunit of the enzyme. In support of this view, isolated domains I and II are competitors for each other, indicating that they are recognized in an equivalent manner by the AspRS subunits. The likely candidate for the interaction with the conserved P2 and P4 motives is the N-terminal extension of AspRS. While this extension is important but not essential for tRNA binding,13,29 it is mandatory for mRNAAspRS recognition.11 As in tRNAAsp, its interaction with P2 and P4, the helical parts of the anticodon arm mimics, likely acts by increasing the global affinity in the complex, but specific interactions cannot be excluded. Thus, it is tempting to propose that the primordial biological function of the extension lies in the regulation of the yeast AspRS. Moreover, since the extension is present also in other eukaryal AspRSs, and in modified versions in eukaryal class IIb synthetases,13 one may wonder whether the mechanisms deciphered for yeast AspRS regulation11,12 are of more general occurrence in the eukaryal synthetase world. As to other structure–function relationships, this work raises a number of questions. The first one concerns the asymmetric nature of the two domains in mRNAAspRS that contact the symmetric structure of AspRS. In particular, the possibility that subdomain Ia harboring the aspartate identity determinants has the primordial role in the regulation mechanism and subdomain IIa, which is an abbreviated mimic, has only an ancillary function, remains unsolved. Second, the anticodon branch mimic in subdomain Ia contains a pseudo-knot. Pseudo-knots in the tRNA world are usually found in the amino acid accepting branch of viral tRNAlike structures. Here, we have the first example of the occurrence of a pseudo-knot in an anticodon branch. Third, the tRNA mimicry in mRNAAspRS points to the presence of an intron-like bulge in the anticodon-like loop in subdomain Ia. This “intronic” sequence could have an essential role in controlling formation of P5, in other words that of the pseudo-knot. Our probing data suggest the existence of both open and closed pseudo-knot

The anticodon-like structures in subdomains Ia and IIa are conserved in yeast species Comparison of known mRNAAspRS sequences from four Saccharomyces species (phylogenetically close to S. cerevisiae) show strong conservation in their coding sequences (Figure 1(b)). For all of them, it is possible to draw a model of secondary structure with an anticodon-like loop containing the AUG initiation codon (Figure 7). This anticodon-like loop always includes a U33-like residue (U2) and 4 nt (G3, U4, C5 and C 7) mimicking aspartate identity determinants in tRNAAsp; namely, the 34GUC36 anticodon and C38. Slight variations appear in P4 of subdomain Ia, with the K3GUGA1 sequence in S. cerevisiae becoming K3ACGA1 in the rest of the Saccharomyces species. Nevertheless, this replacement conserves base-pairing with the UUGU/C sequence at the 3 0 end of the stem. The pseudo-knot found in S. cerevisiae is also present. Finally, despite the weak sequence conservation in the 5 0 UTR, the motif resulting from the long-range interaction UUGU paired to GUGA (forming P2) is strictly conserved but the length of connector C1 in the 5 0 UTR, varies between 12 nt and 19 nt. We are aware that it is difficult to conclude about nucleotide sequence conservations when localized in an ORF. Indeed, one could wonder if these conservations are important for the mRNA structure or for the encoded amino acid sequence. Here, variations in the ORF sequence are compensated by structure maintenance, and the presence of the strictly conserved UUGU/C sequence in the 5 0 UTR is indicative of important RNA structural constraints in the 5 0 extremity of the messengers. If the 1AUGUC5 pentanucleotide within the anticodonlike loop with the AUG initiation codon overlapping the GUC aspartate anticodon is strictly conserved in all yeast species, this sequence is absent from other eukaryotes and makes the search for structural conservations difficult.

Conclusions and Perspectives This work describes a structure of the regulatory region of yeast mRNAAspRS of modular and quasisymmetric architecture that is self-consistent with structural probing experiments, phylogenetic comparisons and computer modeling. This structure can be compared to that present in E. coli mRNA,ThrRS which binds ThrRS,8 where recognition by the synthetase necessitates also two independent RNA domains mimicking the anticodon-loop of tRNAThr.24 The crystal structure

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

627

conformations, but the way by which this molecular switch would be activated remains an open question. The function of the closed pseudo-knot appears established in allowing correct presentation of the aspartate identity determinants to AspRS. Given the similarities with tRNAAsp, the anticodon-like loop in mRNAAspRS should be recognized by the OB-fold anticodon-binding module of AspRS in a way similar to that of the canonical tRNAAsp anticodon loop.30 This view is supported at the phenomenological level by the fact that AspRS1–207 binds the mRNA with the same Kd as the full-length AspRS11 and at the structural level by the modeling of the anticodon-loop mimic in subdomain Ia that strictly superimposes on the tRNAAsp anticodon loop, as occurs in the complex with AspRS. Direct demonstration awaits crystallographic evidences.

reaction in an appropriate buffer containing 50 mM borate–NaOH (pH 8.0), 5 mM magnesium acetate (Merck, Darmstadt, Germany), 15 mM potassium acetate (Merck, Darmstadt, Germany), 5 mM b-mercaptoethanol, 3.2 ng/ml of total yeast tRNA (Boerhinger, Mannheim, Germany). Dimethyl sulfate (DMS) modifications of N1 adenine and N3 cytosine were performed by adding 0.25–2% (w/ v) of DMS (Acros Organics, Geel, Belgium) on 0.5 mg of renatured RNA (as described above), in 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 150 mM KCl, 5mM b-mercaptoethanol and 3.2 ng/ml of total yeast tRNA. Reactions were incubated for 20 min at 30 8C and stopped by ethanolprecipitation.

Materials and Methods Proteins and RNAs Native AspRS and AspRS1–207 (N-terminal extension and anticodon binding domain) have been cloned and purified as described.11 The DNA sequences encoding the mRNAAspRS fragments and their variants were expressed in pUC119 containing the bacteriophage T7 RNA polymerase promoter sequence.11 Mutated mRNAs were obtained by PCR as described by Stratagene (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA). Mutations and deletions were verified by complete sequencing of DNA. Plasmids coding for the different mRNA variants were digested with EcoRI, HaeIII (New England BioLabs, United Kingdom) for full-length and 3 0 shortened mRNA variants, respectively, and used in run-off transcription with bacteriophage T7 RNA polymerase as described.31 Transcripts were purified on a UnoQ column (BioRad, Hercule, CA). mRNAs, used in band shift experiments were internally labeled, by reducing CTP concentration to 150 mM and adding 10 mCi of [a32P]CTP per 1 mg of template DNA in the transcription mixture. Radioactive transcripts were purified on a Nap-5TM desalting column (Amersham Bioscience, Sweden). Notice that all RNA constructs were purified under native conditions to avoid damage of their initial 5 0 –3 0 transcription-induced structure. Because the solubility properties of the RNAs are dependent on the nature of the buffer, it was impossible to test some of the molecules deleted at their 5 0 or 3 0 extremities. It was also difficult to establish unambiguous protein footprints on the mRNA by chemical probing. Only patterns detected with enzymatic probes were reliable for determining the RNA neighborhood in contact with the synthetase. Structural probing and footprint experiments Chemical modifications of RNA For carbodiimide modification of N3 uridine and N1 guanine (CMCT, Merck, Schuchardt, Germany), 0.5 mg of mRNA was re-natured in 20 ml of water, by heating at 65 8C for 1 min, cooling for 1 min on ice and for 10 min at room temperature. CMCT (40–640 mg) was added to the

Enzymatic probing and footprinting Re-natured RNA (0.04 mg/ml) was incubated in 25 mM potassium phosphate (pH 7.5) (Fluka, Buchs, Switzerland), 75 mM KCl, 10 mM MgCl2 alone or with 8 mM AspRS (footprint experiments) for 10 min on ice. RNase V1 (cuts specifically structured regions; USB Corporation, Cleveland, OH) and RNase T2 (cuts single-stranded regions, Invitrogen, Carlsbad, CA) were used in a range of concentrations varying from 0.02 to 0.1 unit per 25 ml reaction. Reactions were stopped by adding one volume of 0.6 mM sodium acetate (Merck, Darmstadt, Germany), 20 mM EDTA and 6 ng/ml of total yeast tRNA, followed by a phenol/chloroform-extraction and ethanolprecipitation.

Assignments of cleavage positions Cleaved/modified RNAs were revealed by primer extension as described.32 Labeled oligonucleotides were chosen to cover nucleotides K38 to C250. RNA was then hydrolyzed by heating the mixture for 3 min at 95 8C in 1 M NaOH, 1 mM EDTA followed by a 60 min incubation at 42 8C. Reverse transcribed DNA was then ethanolˇ erenkov counts were loaded precipitated and 20,000 C onto a denaturing (8 M urea) 12% polyacrylamide gel (acrylamide/bisacrylamide 19/1, w/w) and run for 120 min at 75 W in TBE (89 mM Tris–borate, 2 mM EDTA, pH 8.3). For details on structural probing and footprinting experiments, see Giege´ et al.14

Electrophoretic gel mobility-shift assay ˇ erenkov counts of labeled RNA were A total of 20,000 C heated for 2 min in water at 95 8C, and cooled to room temperature, before addition of 10 mM MgCl2, 50 mM potassium phosphate buffer (pH 7.2), 150 mM KCl, 10% (w/v) glycerol and 40–160 ng/ml of Xenopus laevis 5 S rRNA. AspRS at concentrations varying from 15 nM to 4000 nM was added and mixtures were incubated for 20 min on ice. Bound and unbound labeled RNA molecules were separated by electrophoresis (90 min at 140 V) onto a 6% (w/v) polyacrylamide gel (TBE 1) at 4 8C. Signal analysis and quantification were done on a Fuji Bioimager Bas2000 with Work Station. Quantification of unbound RNA allowed determination of the Kd values corresponding to the AspRS concentration necessary to shift half of the RNA. Each Kd value corresponds to an average of three independent experiments and errors were estimated to be less than 25%.

628

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

Molecular modeling

5. Stelzl, U., Zengel, J. M., Tovbina, M., Walker, M., Nierhaus, K. H., Lindahl, L. & Patel, D. J. (2003). RNAstructural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon. J. Biol. Chem. 278, 28237–28245. 6. Ryckelynck, M., Giege´, R. & Frugier, M. (2005). tRNAs and tRNA mimics as cornerstones of aminoacyl-tRNA synthetases regulations. Biochimie, 87, 835–845. 7. Ibba, M. & So¨ll, D. (2000). Aminoacyl-tRNA synthesis. Annu. Rev. Biochem. 69, 617–650. 8. Romby, P. & Springer, M. (2003). Bacterial translational control at atomic resolution. Trends Genet. 19, 155–161. 9. Li, B., Vilardell, J. & Warner, J. R. (1996). An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness. Proc. Natl Acad. Sci. USA, 93, 1596–1600. 10. White, S. A., Hoeger, M., Schweppe, J. J., Shillingford, A., Shipilov, V. & Zarutskie, J. (2004). Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript. RNA, 10, 369–377. 11. Frugier, M. & Giege´, R. (2003). Yeast aspartyl-tRNA synthetase binds specifically its own mRNA. J. Mol. Biol. 331, 375–383. 12. Frugier, M., Ryckelynck, M. & Giege´, R. (2005). tRNAbalanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway. EMBO Rep. 6, 860–865. 13. Frugier, M., Moulinier, L. & Giege´, R. (2000). A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding. EMBO J. 19, 2371–2380. 14. Giege´, R., Helm, M. & Florentz, C. (2001). Classical and usual chemical tools for RNA structure probing. In RNA (So¨ll, D., Nishimura, S. & Moore, P. B., eds), pp. 71–89, Pergamon/Elsevier, Amsterdam. 15. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucl. Acids Res. 31, 3406–3415. 16. Eriani, G., Cavarelli, J., Martin, F., Dirheimer, G., Moras, D. & Gangloff, J. (1993). Role of dimerization in yeast aspartyl-tRNA synthetase and importance of the class II invariant proline. Proc. Natl Acad. Sci. USA, 90, 10816–10820. 17. Pu¨tz, J., Puglisi, J. D., Florentz, C. & Giege´, R. (1991). Identity elements for specific aminoacylation of yeast tRNAAsp by cognate aspartyl-tRNA synthetase. Science, 252, 1696–1699. 18. Frugier, M., So¨ll, D., Giege´, R. & Florentz, C. (1994). Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetase. Biochemistry, 33, 9912–9921. 19. Ruff, M., Krishnaswamy, S., Boeglin, M., Poterszman, A., Mitschler, A., Podjarny, A. et al. (1991). Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNAAsp. Science, 252, 1682–1689. 20. Quigley, G. J. & Rich, A. (1976). Structural domains of a transfer RNA molecule. Science, 194, 796–806. 21. Ashraf, S. S., Sochacka, E., Cain, R., Guenther, R., Malkiewiewicz, A. & Agris, P. F. (1999). Single atom modification (O/S) of tRNA confers ribosome binding. RNA, 5, 188–194. 22. Abelson, J., Trotta, C. R. & Li, H. (1998). tRNA splicing. J. Biol. Chem. 273, 12685–12688. 23. Sauter, C., Lorber, B., Cavarelli, J., Moras, D. & Giege´, R. (2000). The free yeast aspartyl-tRNA synthetase differs from the tRNAAsp-complexed enzyme by

The three-dimensional model of the mRNAAspRS domain involved in regulation (nucleotides CK38 to G36, and G182 to C197) interacting with AspRS was built using the program MANIP33 and following the RNA modeling strategy developed in-house.34 The three-dimensional model was built using the crystallographic structures of yeast AspRS19 and a model of the enzyme encompassing the additional N-terminal domain.13 The generated model was subjected to restrained least-squares refinement using the programs NUCLIN and NUCLSQ35 to ensure geometry and stereochemistry with allowed distances between interacting atoms and to avoid steric conflicts. Color views were generated with the program VMD†.36

Acknowledgements We thank Jean-Christophe Paillart for advice and stimulating discussion, Christine Brunel for a gift of the 5 S RNA encoding plasmid and Caroline Paulus for technical help, Jean-Luc Souciet for information on yeast genomes, Luc Moulinier and Eric Westhof for support in modeling. This work was supported by grants from Centre National de la Recherche Scientifique (CNRS), Ministe`re de l’Education Nationale de la Recherche et de la Technologie (MENRT), Association pour la Recherche contre le Cancer (ARC), Fondation pour la Recherche Me´dicale (FRM) and Universite´ Louis Pasteur.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2005.09.063 The supplementary material describes chemical probing of S. cerevisiae mRNA(K38,C270).

References 1. Schlax, P. J. & Worhunsky, D. J. (2003). Translational repression mechanisms in prokaryotes. Mol. Microbiol. 48, 1157–1169. 2. Serganov, A., Polonskaia, A., Ehresmann, B. & Patel, D. J. (2003). Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA. EMBO J. 22, 1898–1908. 3. Mathy, N., Pellegrini, O., Serganov, A., Patel, D. J., Ehresmann, C. & Portier, C. (2004). Specific recognition of rpsO mRNA and 16 S rRNA by Escherichie coli ribosomal protein S15 relies on both mimicry and site differentiation. Mol. Microbiol. 52, 661–675. 4. Kraft, A., Lutz, C., Lingenhel, A., Gro¨bner, P. & Piendl, W. (1999). Control of ribosomal protein L1 in mesophilic and thermophilic archaea. Genetics, 152, 1363–1372. † http://www.ks.uiuc.edu/Research/vmd/

mRNA Domain Recognized by Aspartyl-tRNA Synthetase

629

structural changes in the catalytic site, hinge region, and anticodon binding domain. J. Mol. Biol. 299, 1313–1324. Caillet, J., Nogueira, T., Masquida, B., Winter, F., Graffe, M., Dock-Bre´geon, A. C. et al. (2003). The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression. Mol. Microbiol. 47, 961–974. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the ˚ resolution. Science, large ribosomal subunit at 2.4 A 289, 905–920. Leontis, N. B., Stombaugh, J. & Westhof, E. (2002). The non-Watson-Crick base-pairs and their associated isostericity matrices. Nucl. Acids Res. 30, 3497–3531. Torres-Larios, A., Dock-Bregeon, A.-C., Romby, P., Rees, B., Sankaranarayanan, R., Caillet, J. et al. (2002). Structural basis of translational control by Escherichia coli threonyl-tRNA synthetase. Nature Struct. Biol. 9, 343–347. Serganov, A., Ennifar, E., Portier, C., Ehresmann, B. & Ehresmann, C. (2002). Do mRNA and rRNA binding sites of E. coli ribosomal protein S15 share common structural determinants? J. Mol. Biol. 320, 963–978. Ryckelynck, M., Giege´, R. & Frugier, M. (2003). Yeast

tRNAAsp charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase. J. Biol. Chem. 278, 9683–9690. Cavarelli, J. & Moras, D. (1993). Recognition of tRNAs by aminoacyl-tRNA synthetases. FASEB J. 7, 79–86. Frugier, M., Florentz, C., Schimmel, P. & Giege´, R. (1993). Triple aminoacylation specificity of a chimerized transfer RNA. Biochemistry, 32, 14053–14061. Baudin, F., Marquet, R., Isel, C., Darlix, J. L., Ehresmann, B. & Ehresmann, C. (1993). Functional sites in the 5 0 region of human immunodeficiency virus type 1 RNA form defined structural domains. J. Mol. Biol. 229, 382–397. Massire, C. & Westhof, E. (1998). MANIP: an interactive tool for modelling RNA. J. Mol. Graph. Model, 16, 197–205. Masquida, B. & Westhof, E. (2005). Modeling the architecture of structured RNAs within a modular and hierarchical framework. In Handbook of RNA Biochemistry (Hartmann, R. K., Bindereif, A., Scho¨n, A. & Westhof, E., eds), pp. 536–545, Wiley, London. Westhof, E. (1993). Modelling the three-dimensional structure of ribonucleic acids. J. Mol. Struct. 286, 203–210. Humphrey, W., Dalke, A. & Schulten, K. (1996). VMD—Visual molecular dynamics. J. Mol. Graph. 14.1, 33–38.

24.

25.

26. 27.

28.

29.

30. 31. 32.

33. 34.

35. 36.

Edited by J. Doudna (Received 18 July 2005; received in revised form 19 September 2005; accepted 20 September 2005) Available online 5 October 2005