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Complexes of tRNA and maturation enzymes: shaping up for translation
Complexes of tRNA and maturation enzymes: shaping up for translation Hong Li Several significant structures of transfer ribonucleic acid (tRNA) maturation enzymes complexed with precursor tRNA or fragments thereof have been published recently, providing detailed knowledge of enzyme–tRNA recognition and catalytic strategies. In addition to reinforcing the general principles of RNA–protein interaction, the new structures highlight both the features of composite RNA recognition by multiple enzyme subunits and the pronounced RNA structural flexibility in or near the active site in all cases. These structural principles provide plausible explanations for the exquisite specificity and catalytic power of these enzymes and, in the case of evolutionary adaptation, for the ability of some enzymes to develop novel specificities. Addresses Department of Chemistry and Biochemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA Corresponding author: Li, Hong ([email protected])
Current Opinion in Structural Biology 2007, 17:293–301 This review comes from a themed issue on Nucleic acids Edited by Dinshaw J Patel and Eric Westhof Available online 18th June 2007 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.05.002
Introduction As the adaptor between mRNA and proteins, transfer ribonucleic acid (tRNA) carries out indispensable functions in translation during protein synthesis. In landmark discoveries, its function was theorized by Francis Crick in 1955 and confirmed experimentally in the 1960s [1,2]. Subsequently, tRNA became the first stable RNA viewed by X-ray crystallography [3–5]. High-resolution structures of translating ribosomes trapped in intricately balanced complexes with tRNA have increased our appreciation of this fascinating molecule [6,7]. Despite being relatively small, the tRNA structure holds a ‘‘treasury of stereochemical information’’ [8], and exhibits rich principles of RNA folding [1,9] and RNA–protein interaction [10,11]. Nascent tRNA molecules are not suited for translation. After transcription by RNA polymerases, they must undergo numerous processing and modification steps, which can include removal of introns, trimming of 50 leader and 30 trailer sequences, addition of CCA to 30 www.sciencedirect.com
ends, and several base and backbone modifications [12]. In yeast, it is estimated that more than 60 proteins are responsible for tRNA maturation and produce 3–6 million tRNAs per cell generation [13]. With the exception of the ribozyme ribonuclease (RNase) P [14,15], which catalyzes RNA cleavage reactions using RNA, all tRNA processing and modification enzymes are known to be composed of proteins. Previously characterized structural complexes involving tRNA largely comprise tRNA with aminoacyl-tRNA synthetases and translation factors. Aminoacyl-tRNA synthetases recognize the overall shape of tRNA and distinguish specific tRNAs by features present in anticodons, acceptor stems and extra loops [10,11]. At least two aminoacyl-tRNA synthetases splay anticodon nucleotides out of base stacking for a direct readout [16,17]. Crosssubunit recognition is observed in several synthetase– tRNA complexes [11]. More detailed structural features of tRNA synthetase complexes are reviewed elsewhere [10,11,18] and thus are not covered here. Structural studies on enzymes that mature tRNA have advanced rapidly in the wake of their genomic identification and biochemical characterization ([12,19]; Table 1). Unlike aminoacyl-tRNA synthetases, which exploit entire tRNA topologies [10,11,18], but similar to translation factors [20], most tRNA modification and tRNA processing enzymes target the peripheral regions of the L-shaped tRNA (Figure 1). These enzymes have evolved to provide catalytic power for a variety of chemical reactions at sites often deeply protected by folded precursor tRNA. How are catalytic targets specifically recognized and how are the reactions instigated? Exploration of answers to these questions can be complicated in the context of evolution: homologous enzymes often display significantly different substrate specificities, while their catalytic mechanisms are conserved [12,21,22,23]. This review focuses on selected recent studies that have addressed these aspects of tRNA maturation enzymes. New structural results from these studies illustrate two common themes that are also highlighted herein. One is the modular but coordinated RNA recognition by multiple subunits and the other is RNA structure remodeling. The implications of these structural properties for substrate specificity, catalytic strategy and evolutional adaptation are discussed later.
Intron recognition by splicing endonucleases Intervening sequences (introns) are found in 4–25% of archaeal and eukaryal tRNA genes. Evolutionarily Current Opinion in Structural Biology 2007, 17:293–301
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Table 1 Recently determined crystal structures of tRNA processing and modification complexes. tRNA–protein complex
Enzyme function
Structural characteristics
References and PDB codes
Acceptor binding Class I CCA-adding enzyme–RNA
30 -end addition of CCA
tRNase Z–RNA
30 -end processing
[41,42]; 1SZ1, 1TFY, 1TFW and 2DR2, 2DR7, 2DR8, 2DR9, 2DRA, 2DRB, 2DVI [31]; 2FK6
GatDE–RNA
Conversion of Glu-tRNAGln to Gln-tRNA Gln
Shape recognition of the acceptor stem by a tethered domain, trapped reaction intermediates, acceptor stem expands and contracts, base scrunching Composite recognition of acceptor stem, 30 -end base remodeling Minor groove recognition of acceptor and D-stem, identity elements in acceptor region recognized, 40 A˚ diffusion channel for ammonia Base remodeling for recognition of UU, trapped adenylated-U intermediates Composite recognition of anticodon, base remodeling
[39]; 2DER, 2DET, 2DEU
Composite recognition of anticodon, base remodeling, trapped covalent adduct
[44]; 1Q2R, 1Q2S
Anticodon binding MnmA–RNA TadA–RNA
Conversion of U34 to s4U34
[43]; 2D6F
[35]; 2B3J
TGT–RNA
Adenosine-to-inosine editing of A34 Replace G34 by preQ1
D-loop binding ArcTGT–RNA
Replace G15 by preQ0
Significant remodeling of D-stem, cross-subunit recognition of acceptor terminus
[45]; 1J2B
Removal of introns
Composite recognition of BHB RNA, base remodeling, trapped reaction products
[29]; 2GJW
U55 pseudouridylation
Minor groove recognition of TCC loop, base remodeling, trapped reaction intermediate
[47,48]; 1K8W, 1R3E
Intron binding Splicing endonuclease– RNA T-stem binding TruB–RNA
TGT: tRNA guanine transglycosylase; ArcTGT: archaeosine tRNA guanine transglycosylase.
conserved splicing endonucleases, first identified and isolated from yeast [24,25], act at the first step of splicing and are responsible for the recognition and excision of Figure 1
Schematic of tRNA precursors subjected to the (a) processing and (b) modification processes discussed in the text. The major sites of precursor tRNA recognized by processing and modification enzymes (colored blocks) are indicated. These include the TCC stem-loop, anticodon and acceptor stem-loop and, for some precursors, the intron. Current Opinion in Structural Biology 2007, 17:293–301
these introns from precursor tRNA after transcription. A key feature of splicing endonuclease function is organism-specific substrate recognition [22]. A splicing endonuclease recognizes a folded RNA motif (rather than a base sequence) that incorporates the two intron–exon junctions. In archaea, most intron–exon junctions are found in two three-nucleotide bulges separated by four base pairs (the bulge-helix-bulge [BHB] motif). Variations of the canonical BHB motif are observed in archaeal substrates and can be clearly correlated with archaeal phyla [21,26,27]. In eukaryotes, introns are invariably located one base 30 to the anticodon. In this case, the enzyme exploits a ‘ruler mechanism’ and locates the intron–exon junctions by measurement [28]. The composition of endonuclease subunits is also phylogenetically correlated. The splicing endonucleases identified so far are classified into a4, a2, (ab)2 and abgd families. The first three families are found in archaea and the abgd family appears only in eukaryotes. Regardless of the number of subunits, the enzymes contain four homologous units — two catalytic and two structural. The two catalytic units are symmetrically placed, with the active sites 28 A˚ diagonally apart, through a cyclic interaction among the units (structural!catalytic!structural!catalytic). This enzyme architecture is absolutely essential for the recognition of the two cleavage sites and, more www.sciencedirect.com
Complexes of tRNA and maturation enzymes Li 295
Figure 2
Overview of recent selected examples of tRNA–maturation enzyme complexes. Proteins are colored green and blue for the two subunits in (a–c), and green, blue and cyan for separate domains in (d). The bound RNAs are colored orange (backbone) and red (bases). (a) The splicing endonuclease binds to the intron–exon junction during intron removal. (b) tRNase Z binds to the tRNA acceptor stem during 30 -end processing. (c) TadA and (d) MnmA bind to anticodon loops during adenosine-to-inosine editing and sulfuration, respectively. www.sciencedirect.com
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importantly, for the stabilization of the transition state during catalysis. A recently determined crystal structure of a dimeric (a2) splicing endonuclease complexed with a BHB RNA (Figure 2a) revealed that two catalytic units interact with one cleavage site in a surprisingly coordinated fashion [29]. One catalytic unit holds the bulge, which is cleaved by the other (Figure 3a). The interdependence of the two catalytic units prevents the enzyme from binding and cleaving RNA promiscuously, and explains the critical importance of enzyme assembly for the control of catalytic activity.
Figure 3
Recognition of the three-nucleotide bulge and excision of the phosphodiester bond of the second bulge nucleotide are made possible by the inherent structural flexibility of the bulge. Remarkably, all three bulge nucleotides are extruded, with only the first bulge nucleotide interacting significantly with the endonuclease (Figure 4a). The nucleobase of this nucleotide is sandwiched by a pair of arginine residues from the opposing catalytic subunit (Figure 5a). The critical importance and nature of the conserved cation–p interaction explains well why the nucleobases of the bulge nucleotides are not strictly conserved [29]. The phosphate backbone of the bulge is significantly bent to complement the relatively rigid active site of the enzyme (see Figure 5a). This recognition mechanism also assists catalysis, therefore, because it enables the three atoms involved in the phosphotransfer reaction to be aligned in a near attack conformation that is otherwise energetically unfavorable [29,30] (see Figure 5a).
Acceptor recognition by tRNase Z The 30 end of tRNA is the site of amino acid attachment. The precise length and placement of nucleotide functional groups in this region play critical roles in the aminoacylation process. Recent structural studies on an acceptor-processing enzyme highlight a new principle of upper stem recognition by composite binding sites [31,32]. tRNase Z is a universally conserved dimeric enzyme that endonucleolytically trims the 30 end of tRNA. Similar to splicing endonucleases, tRNA Z exhibits sequenceindependent but organism-specific substrate specificity for precursor tRNA. For instance, Thermotoga maritima tRNase Z cleaves after the CCA sequence, some archaeal enzymes cleave after CC and the Bacillus subtilis enzyme cleaves strictly after the discriminator nucleotide immediately before the CCA sequence [23]. The crystal structure of B. subtilis tRNase Z (with a catalytic histidine to alanine mutation) bound to a pre-tRNAThr that contains 30 extensions [31] shows that both enzyme monomers recognize the precursor tRNA in a coordinated fashion. A domain attached to the catalytic domain of one subunit rests on the platform formed by the T-stem, but it is the adjacent catalytic subunit that interacts with Current Opinion in Structural Biology 2007, 17:293–301
Composite recognition of tRNA motifs by maturation enzymes. Residues that interact with tRNA are shown as stick models and are colored by subunit (green, subunit 1; blue, subunit 2). (a) Two catalytic units of the dimeric splicing endonuclease coordinately interact with the two splice junctions; the site stabilized by the green subunit is cleaved by the blue subunit and vice versa. (b) The tRNAthr precursor is primarily bound by the green subunit of tRNase Z while it is cleaved by the blue subunit. (c) Two subunits of TadA recognize the anticodon stem and loop.
the first base pair (1 and 72) and discriminator base 73 (see Figures 2b and 3b). U73 of tRNAThr unstacks from the acceptor stem to enter the active site of the enzyme, where it is co-stabilized by both subunits (see Figure 4b). www.sciencedirect.com
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Figure 4
A tyrosine residue from one subunit and a phenylalanine residue from the other sandwich the U73 nucleobase (see Figure 3b). Unfortunately, no structural information is available for the 30 extension nucleotides, which prevents detailed analysis of the catalytic mechanism. Based on the composite RNA-binding site and the apparent flexibility of the 30 end of tRNA, however, it has been proposed that subtle differences in the dimeric arrangement and accessibility of the active site could account for the different effects of CCA on the enzymes [23]. These differences cannot explain why some enzymes can cleave both CCA-less and CCA-plus substrates, however.
Anticodon recognition by deaminase TadA and thiouridylase MnmA Anticodons play an indispensable role in codon recognition during protein translation. The structures of the anticodon of tRNA both when free [5] and when bound to ribosomes [7] reflect their ability to directly read the cognate codons in mRNA. Numerous chemical modifications are found in this region of many tRNAs after intron removal and 50 /30 trimming [33]. Recently published examples of two anticodon modifying enzymes discussed below illustrate a general mechanism for how enzymes access anticodon loop nucleotides. Both of these enzymes recognize anticodon-specific features, especially nucleotide 34, by flipping out nucleobases. TadA is a specific adenosine-to-inosine editing enzyme for wobble position 34 of bacterial tRNAArg2 [34]. This modification enables tRNA to recognize CGA/U/C codons. TadA is relatively small and functions as a homodimer [34]. In the crystal structure of Staphylococcus aureus TadA with anticodon stem-loop RNA [35], two RNA anticodon loops are bound symmetrically to the TadA dimer, in which each RNA loop is contacted by both subunits of the dimer (see Figure 2c). In particular, the dimeric enzyme coordinately recognizes the non-Watson–Crick base pair between nucleotides 32 and 38 (C32–A38) that caps the anticodon loop by forming a network of hydrogen bonds with both subunits (see Figure 3c). A prominent role for this base pair in stabilizing the anticodon hairpin and in conferring tRNA specificity has been gradually recognized [36,37]. The cross-subunit stabilization of the anticodon loop by the S. aureus TadA homodimer agrees well with the fact that yeast tRNA is edited by the heterodimeric enzyme Tad2p–Tad3p, with each of the subunits being essential [38]. Base splaying is a common feature of tRNA–maturation enzyme complexes. (a) The splicing endonuclease flips all three bulge nucleotides in the BHB motif. (b) tRNase Z flips nucleotide 73 immediately preceding the target phosphodiester bond. (c) TadA and (d) MnmA flip target nucleotide 34 for complete access to the catalytic target.
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A remarkable feature observed in the TadA–tRNAArg2 complex is the significant remodeling of anticodon nucleotides. The nucleobases of 33, 34, 35 and 37 are splayed outward to interact with the active site pocket of the enzyme (see Figure 4c). Both hydrogen-bonding and cation–p stacking interactions are used by the enzyme to stabilize the splayed bases. G36 is the only anticodon Current Opinion in Structural Biology 2007, 17:293–301
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Figure 5
Exploration of the structural flexibility of tRNA by maturation enzymes for catalytic power. In each panel, the right side shows the reaction catalyzed by the enzyme and the left side displays detailed structures at the enzyme active sites. (a) The splicing endonuclease catalyzes an intramolecular phosphotransfer reaction via a pentacordinated intermediate in an in-line conformation. The two nucleotides flanking the scissile phosphate form the in-line conformation at the enzyme active site in preparation for bond breakage. Note that the adjacent 20 -OH group was modeled into the crystal structure lacking this group. (b) Anticodon nucleotide A34 (Neb34 in the structure) is well placed to interact with a catalytic water, the catalytic zinc and the essential glutamate residue, Glu55, of TadA. (c) The intermediate of adenylated uridine in the active site of MnmA is placed for catalysis. Cys199 is suggested to be the sulfur acceptor from a sulfur relay system that is eventually transferred to the C2 atom of U34.
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nucleotide that points to the interior of the anticodon loop, although its nucleobase is also stabilized by hydrogen bonds with the enzyme. The catalytic target nucleotide, Neb34 (nebularine, a nucleobase analog of adenosine, was used at position 34 for co-crystallization), is the most buried nucleotide and forms several interactions with the catalytic groups (see Figure 5b). However, the lack of a 6-amino group of Neb34 is thought to prevent the enzyme from forming the prehydrolytic deamination state [35]. The network of interactions formed by the splayed anticodon nucleotides with the rigid enzyme active site and water molecules is theorized to contribute to the strength and specificity of the TadA–RNA interaction [35]. MnmA is a bacterial thiouridylase that also modifies anticodon nucleotide 34 [39]. MnmA is responsible for the conversion of U34 to s4U34 in tRNAGlu, tRNALys and tRNAGln, all of which contain UUN anticodon sequences [1]. MnmA uses an elaborate two-step mechanism to sulfurate U34 via an adenylated U34 intermediate [39]. Therefore, MnmA must recognize and fully access the unique tandem uridine nucleotides in the anticodon. MnmA binds to tRNA as a monomer, but with outspread protein domains (see Figure 2d). The protein–RNA interface extends from the anticodon loop to the bottom of the D-stem. The central domain, a glycine-rich loop in particular, reaches up to the D-stem and facilitates minor groove recognition. The catalytic domain and an EF-Tu-like domain nestle in the anticodon region, in which the two uridine nucleotides are most extensively stabilized by hydrogen bonds. This stabilization is, once again, facilitated by extruded bases. The catalytic target uridine is completely extruded from the stacked anticodon into the deep pocket of the catalytic domain (see Figures 2d and 4d). The other two anticodon nucleotides, however, remain partially stacked in the enzyme (see Figures 2d and 4d). The resulting active site, especially after adenylation of U34, forms a closed chamber in preparation for the sulfur-transfer reaction (see Figure 5c).
Conclusions Two overriding themes evident from these structural studies expand characteristics uncovered in other tRNA complex studies: significant flexibility of RNA at the processing or modifying sites, and composite (or crosssubunit) RNA recognition by multiple enzyme subunits. Structural remodeling seems to be the most direct method for enzymes to access folded RNA bases. Besides those described here and aminoacyl-tRNA synthetases, other tRNA maturation enzymes also adopt this strategy to access their catalytic targets (Table 1). These include class I and II tRNA CCA-adding enzymes [40,41,42], amidation transferase GatDE [43], G15 and G34 transglycosylases [44–46] and U55 pseudouridylase [46–48]. Together, these examples suggest that many modifying www.sciencedirect.com
and processing enzymes exploit the intrinsic flexibility of tRNA to gain catalytic power towards specific targets. In addition, the structural flexibility of RNA provides a means for some enzymes to achieve organism-specific RNA recognition. Whereas relatively rigid active sites are intolerant of slight variations in the target bases of RNA substrates, more elastic active sites of the homologous enzymes may permit the variations by ‘scrunching’ the bases. An extraordinary example of the scrunching model was elegantly demonstrated for the class I CCA-adding enzyme. The three nucleotides at the 30 end of a tRNA are added in a nucleotide-wise fashion by the single active site of the CCA-adding enzyme [41,42]. It remains to be seen whether the patterns of substrate specificity for splicing endonucleases and tRNase Z can be explained by similar models. Subunit composition is another facet to the mechanism of tRNA maturation enzymes. Regardless of the number of catalytic target sites, the enzymes discussed here have multiple subunits or domains that form composite binding sites for RNA substrates. By jointly recognizing RNA features, composite binding ensures substrate specificity. In general, oligomerization enhances binding affinity by increasing the amount of surface that contacts the substrate and can regulate enzyme activity according to the intracellular enzyme concentration. Furthermore, oligomerization affords the flexibility for individual subunits to specialize, potentially enabling functional connections with other cellular machines. The evolutionary histories of the enzymes discussed here have already begun to shed light on this hypothesis [49].
Looking into the future tRNA maturation enzymes catalyze some of the most elaborate reactions known, which makes catalytic strategy a fascinating counterpart to the study of tRNA recognition. What are the functional groups in tRNA and enzymes that stabilize reaction intermediates, and how are these related to the intrinsic flexibility of RNA and enzyme organization? The excitement about elucidating these mechanisms is now well placed.
Acknowledgements The author is grateful for scholarly discussions with Eric Westhof, Anita Marchfelder, Dieter So¨ll, Robert Reeves, Christopher Trotta, Salvatore San Paolo, Michaeal P Terns, Rebecca M Terns, Wei Yang, Kate Calvin and Song Xue. The author thanks the National Institutes of Health (R01 GM66958-01) and National Science Foundation (MCB-0517300) for financial support.
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34. Wolf J, Gerber AP, Keller W: TadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J 2002, 21:3841-3851. 35. Losey HC, Ruthenburg AJ, Verdine GL: Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA. Nat Struct Mol Biol 2006, 13:153-159. The tRNA adenosine deaminase TadA is an essential bacterial enzyme responsible for adenosine-to-inosine editing of the wobble base (A34) of tRNAArg2. The edited tRNA is capable of reading CUA/U/C codons. The co-crystal structure of S. aureus TadA bound to the anticodon stem-loop of tRNAArg2 bearing nebularine, a non-hydrolyzable adenosine analog, www.sciencedirect.com
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shows a network of interactions between the enzyme and the RNA. Anticodon nucleotides are splayed in the complex to facilitate the specificity of recognition and catalysis. This recognition is facilitated by dimerized enzyme subunits and explains the specificity of the enzyme for tRNAArg2. 36. Auffinger P, Westhof E: Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes. J Mol Biol 1999, 292:467-483. 37. Olejniczak M, Uhlenbeck OC: tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition. Biochimie 2006, 88:943-950. 38. Gerber AP, Keller W: An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science 1999, 286:1146-1149. 39. Numata T, Ikeuchi Y, Fukai S, Suzuki T, Nureki O: Snapshots of tRNA sulphuration via an adenylated intermediate. Nature 2006, 442:419-424. 20 -thio modification of uridine at the first anticodon position (U34) of tRNAGlu, tRNALys and tRNAGln is phylogenetically conserved. This is carried out by MnmA thiouridylase via two enzymatic steps and an adenylated nucleotide intermediate. The co-crystal structures of E. coli MnmA and tRNAGlu in three functional states illustrate one principle of tRNA anticodon stem-loop recognition, similar to what is observed for TadA; anticodon nucleotide U34 is splayed outwards to reach the enzyme active site for modification. Active site rearrangement enables identification of catalytically important residues. Two possible catalytic mechanisms are proposed. 40. Xiong Y, Steitz TA: A story with a good ending: tRNA 30 -end maturation by CCA-adding enzymes. Curr Opin Struct Biol 2006, 16:12-17. 41. Xiong Y, Steitz TA: Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 2004, 430:640-645. The CCA-adding enzyme is responsible for the addition of the 30 -terminal CCA sequence to tRNA. Crystal structures of the Archaeoglobus fulgidus tRNA nucleotidyltransferase (a class I CCA-adding enzyme) in complex with yeast tRNAPhe at low resolution and in three distinct complexes with the acceptor stem at better than 3.4 A˚ resolution offer significant insights into how CCA is polymerized without a nucleic acid template. The remarkable series of images captured at each enzymatic step — after the addition of C74, after the addition of C75 and after the addition of A76 — showed how the enzyme can switch specificity from CTP to ATP and that the enzyme active site is progressively altered by the elongating 30 end of the tRNA. However, the lack of the TCC loop in the model acceptor stem, which contacts the RNA-binding tail domain, could affect the precise ways in which the RNA and the active site residues rearrange in these crystals. 42. Tomita K, Ishitani R, Fukai S, Nureki O: Complete crystallographic analysis of the dynamics of CCA sequence addition. Nature 2006, 443:956-960.
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This work describes six distinct complexes of the A. fulgidus CCA-adding enzyme with a TCC-loop-containing minihelix derived from Thermotoga maritima tRNAPhe, and with or without incoming nucleotides. Significant protein and RNA dynamics were observed at different polymerizing stages; however, these were somewhat different from those described in [41]. The acceptor stem expands and contracts in the first polymerization step. The subsequent steps are similar to those described in [41] in that the primer nucleotides splay out of the way so that the incoming NTP can be accommodated in the catalytic cleft. The sidechain of an important arginine residue was observed in different conformations depending on the polymerization stage; this is believed to achieve CTP/ATP discrimination. 43. Oshikane H, Sheppard K, Fukai S, Nakamura Y, Ishitani R, Numata T, Sherrer RL, Feng L, Schmitt E, Panvert M et al.: Structural basis of RNA-dependent recruitment of glutamine to the genetic code. Science 2006, 312:1950-1954. The amidotransferase GatDE is a heterodimeric enzyme that converts the misacylated Glu-tRNAGln by amidation to Glu-tRNAGlu as a unique charging process in archaea. GatDE uses separate subunits to produce ammonia, to phosphorylate the g-carboxyl group of the tRNA-bound glutamate and to convert phosphoglutamate to glutamine. This work describes a co-crystal structure of Methanothermobacter thermautotrophicus GatDE in complex with tRNAGln at 3.2 A˚ resolution. The structure reveals a surprising method of tRNAGln recognition that is independent of anticodon. The shape and the tRNAGln-specific bases of the acceptor stem are recognized by the enzyme. The structure also sheds light on the mechanism of ammonia recruitment through an 40 A˚ tunnel and repeating hydrogen bonding. 44. Xie W, Liu X, Huang RH: Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat Struct Biol 2003, 10:781-788. 45. Ishitani R, Nureki O, Nameki N, Okada N, Nishimura S, Yokoyama S: Alternative tertiary structure of tRNA for recognition by a posttranscriptional modification enzyme. Cell 2003, 113:383-394. 46. Ferre-D’Amare AR: RNA-modifying enzymes. Curr Opin Struct Biol 2003, 13:49-55. 47. Hoang C, Ferre-D’Amare AR: Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme. Cell 2001, 107:929-939. 48. Pan H, Agarwalla S, Moustakas DT, Finer-Moore J, Stroud RM: Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit. Proc Natl Acad Sci USA 2003, 100:12648-12653. 49. Paushkin SV, Patel M, Furia BS, Peltz SW, Trotta CR: Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 30 end formation. Cell 2004, 117:311-321.
Current Opinion in Structural Biology 2007, 17:293–301
Current Opinion in Structural Biology 2007, 17:293–301 This review comes from a themed issue on Nucleic acids Edited by Dinshaw J Patel and Eric Westhof Available online 18th June 2007 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.05.002
Introduction As the adaptor between mRNA and proteins, transfer ribonucleic acid (tRNA) carries out indispensable functions in translation during protein synthesis. In landmark discoveries, its function was theorized by Francis Crick in 1955 and confirmed experimentally in the 1960s [1,2]. Subsequently, tRNA became the first stable RNA viewed by X-ray crystallography [3–5]. High-resolution structures of translating ribosomes trapped in intricately balanced complexes with tRNA have increased our appreciation of this fascinating molecule [6,7]. Despite being relatively small, the tRNA structure holds a ‘‘treasury of stereochemical information’’ [8], and exhibits rich principles of RNA folding [1,9] and RNA–protein interaction [10,11]. Nascent tRNA molecules are not suited for translation. After transcription by RNA polymerases, they must undergo numerous processing and modification steps, which can include removal of introns, trimming of 50 leader and 30 trailer sequences, addition of CCA to 30 www.sciencedirect.com
ends, and several base and backbone modifications [12]. In yeast, it is estimated that more than 60 proteins are responsible for tRNA maturation and produce 3–6 million tRNAs per cell generation [13]. With the exception of the ribozyme ribonuclease (RNase) P [14,15], which catalyzes RNA cleavage reactions using RNA, all tRNA processing and modification enzymes are known to be composed of proteins. Previously characterized structural complexes involving tRNA largely comprise tRNA with aminoacyl-tRNA synthetases and translation factors. Aminoacyl-tRNA synthetases recognize the overall shape of tRNA and distinguish specific tRNAs by features present in anticodons, acceptor stems and extra loops [10,11]. At least two aminoacyl-tRNA synthetases splay anticodon nucleotides out of base stacking for a direct readout [16,17]. Crosssubunit recognition is observed in several synthetase– tRNA complexes [11]. More detailed structural features of tRNA synthetase complexes are reviewed elsewhere [10,11,18] and thus are not covered here. Structural studies on enzymes that mature tRNA have advanced rapidly in the wake of their genomic identification and biochemical characterization ([12,19]; Table 1). Unlike aminoacyl-tRNA synthetases, which exploit entire tRNA topologies [10,11,18], but similar to translation factors [20], most tRNA modification and tRNA processing enzymes target the peripheral regions of the L-shaped tRNA (Figure 1). These enzymes have evolved to provide catalytic power for a variety of chemical reactions at sites often deeply protected by folded precursor tRNA. How are catalytic targets specifically recognized and how are the reactions instigated? Exploration of answers to these questions can be complicated in the context of evolution: homologous enzymes often display significantly different substrate specificities, while their catalytic mechanisms are conserved [12,21,22,23]. This review focuses on selected recent studies that have addressed these aspects of tRNA maturation enzymes. New structural results from these studies illustrate two common themes that are also highlighted herein. One is the modular but coordinated RNA recognition by multiple subunits and the other is RNA structure remodeling. The implications of these structural properties for substrate specificity, catalytic strategy and evolutional adaptation are discussed later.
Intron recognition by splicing endonucleases Intervening sequences (introns) are found in 4–25% of archaeal and eukaryal tRNA genes. Evolutionarily Current Opinion in Structural Biology 2007, 17:293–301
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Table 1 Recently determined crystal structures of tRNA processing and modification complexes. tRNA–protein complex
Enzyme function
Structural characteristics
References and PDB codes
Acceptor binding Class I CCA-adding enzyme–RNA
30 -end addition of CCA
tRNase Z–RNA
30 -end processing
[41,42]; 1SZ1, 1TFY, 1TFW and 2DR2, 2DR7, 2DR8, 2DR9, 2DRA, 2DRB, 2DVI [31]; 2FK6
GatDE–RNA
Conversion of Glu-tRNAGln to Gln-tRNA Gln
Shape recognition of the acceptor stem by a tethered domain, trapped reaction intermediates, acceptor stem expands and contracts, base scrunching Composite recognition of acceptor stem, 30 -end base remodeling Minor groove recognition of acceptor and D-stem, identity elements in acceptor region recognized, 40 A˚ diffusion channel for ammonia Base remodeling for recognition of UU, trapped adenylated-U intermediates Composite recognition of anticodon, base remodeling
[39]; 2DER, 2DET, 2DEU
Composite recognition of anticodon, base remodeling, trapped covalent adduct
[44]; 1Q2R, 1Q2S
Anticodon binding MnmA–RNA TadA–RNA
Conversion of U34 to s4U34
[43]; 2D6F
[35]; 2B3J
TGT–RNA
Adenosine-to-inosine editing of A34 Replace G34 by preQ1
D-loop binding ArcTGT–RNA
Replace G15 by preQ0
Significant remodeling of D-stem, cross-subunit recognition of acceptor terminus
[45]; 1J2B
Removal of introns
Composite recognition of BHB RNA, base remodeling, trapped reaction products
[29]; 2GJW
U55 pseudouridylation
Minor groove recognition of TCC loop, base remodeling, trapped reaction intermediate
[47,48]; 1K8W, 1R3E
Intron binding Splicing endonuclease– RNA T-stem binding TruB–RNA
TGT: tRNA guanine transglycosylase; ArcTGT: archaeosine tRNA guanine transglycosylase.
conserved splicing endonucleases, first identified and isolated from yeast [24,25], act at the first step of splicing and are responsible for the recognition and excision of Figure 1
Schematic of tRNA precursors subjected to the (a) processing and (b) modification processes discussed in the text. The major sites of precursor tRNA recognized by processing and modification enzymes (colored blocks) are indicated. These include the TCC stem-loop, anticodon and acceptor stem-loop and, for some precursors, the intron. Current Opinion in Structural Biology 2007, 17:293–301
these introns from precursor tRNA after transcription. A key feature of splicing endonuclease function is organism-specific substrate recognition [22]. A splicing endonuclease recognizes a folded RNA motif (rather than a base sequence) that incorporates the two intron–exon junctions. In archaea, most intron–exon junctions are found in two three-nucleotide bulges separated by four base pairs (the bulge-helix-bulge [BHB] motif). Variations of the canonical BHB motif are observed in archaeal substrates and can be clearly correlated with archaeal phyla [21,26,27]. In eukaryotes, introns are invariably located one base 30 to the anticodon. In this case, the enzyme exploits a ‘ruler mechanism’ and locates the intron–exon junctions by measurement [28]. The composition of endonuclease subunits is also phylogenetically correlated. The splicing endonucleases identified so far are classified into a4, a2, (ab)2 and abgd families. The first three families are found in archaea and the abgd family appears only in eukaryotes. Regardless of the number of subunits, the enzymes contain four homologous units — two catalytic and two structural. The two catalytic units are symmetrically placed, with the active sites 28 A˚ diagonally apart, through a cyclic interaction among the units (structural!catalytic!structural!catalytic). This enzyme architecture is absolutely essential for the recognition of the two cleavage sites and, more www.sciencedirect.com
Complexes of tRNA and maturation enzymes Li 295
Figure 2
Overview of recent selected examples of tRNA–maturation enzyme complexes. Proteins are colored green and blue for the two subunits in (a–c), and green, blue and cyan for separate domains in (d). The bound RNAs are colored orange (backbone) and red (bases). (a) The splicing endonuclease binds to the intron–exon junction during intron removal. (b) tRNase Z binds to the tRNA acceptor stem during 30 -end processing. (c) TadA and (d) MnmA bind to anticodon loops during adenosine-to-inosine editing and sulfuration, respectively. www.sciencedirect.com
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importantly, for the stabilization of the transition state during catalysis. A recently determined crystal structure of a dimeric (a2) splicing endonuclease complexed with a BHB RNA (Figure 2a) revealed that two catalytic units interact with one cleavage site in a surprisingly coordinated fashion [29]. One catalytic unit holds the bulge, which is cleaved by the other (Figure 3a). The interdependence of the two catalytic units prevents the enzyme from binding and cleaving RNA promiscuously, and explains the critical importance of enzyme assembly for the control of catalytic activity.
Figure 3
Recognition of the three-nucleotide bulge and excision of the phosphodiester bond of the second bulge nucleotide are made possible by the inherent structural flexibility of the bulge. Remarkably, all three bulge nucleotides are extruded, with only the first bulge nucleotide interacting significantly with the endonuclease (Figure 4a). The nucleobase of this nucleotide is sandwiched by a pair of arginine residues from the opposing catalytic subunit (Figure 5a). The critical importance and nature of the conserved cation–p interaction explains well why the nucleobases of the bulge nucleotides are not strictly conserved [29]. The phosphate backbone of the bulge is significantly bent to complement the relatively rigid active site of the enzyme (see Figure 5a). This recognition mechanism also assists catalysis, therefore, because it enables the three atoms involved in the phosphotransfer reaction to be aligned in a near attack conformation that is otherwise energetically unfavorable [29,30] (see Figure 5a).
Acceptor recognition by tRNase Z The 30 end of tRNA is the site of amino acid attachment. The precise length and placement of nucleotide functional groups in this region play critical roles in the aminoacylation process. Recent structural studies on an acceptor-processing enzyme highlight a new principle of upper stem recognition by composite binding sites [31,32]. tRNase Z is a universally conserved dimeric enzyme that endonucleolytically trims the 30 end of tRNA. Similar to splicing endonucleases, tRNA Z exhibits sequenceindependent but organism-specific substrate specificity for precursor tRNA. For instance, Thermotoga maritima tRNase Z cleaves after the CCA sequence, some archaeal enzymes cleave after CC and the Bacillus subtilis enzyme cleaves strictly after the discriminator nucleotide immediately before the CCA sequence [23]. The crystal structure of B. subtilis tRNase Z (with a catalytic histidine to alanine mutation) bound to a pre-tRNAThr that contains 30 extensions [31] shows that both enzyme monomers recognize the precursor tRNA in a coordinated fashion. A domain attached to the catalytic domain of one subunit rests on the platform formed by the T-stem, but it is the adjacent catalytic subunit that interacts with Current Opinion in Structural Biology 2007, 17:293–301
Composite recognition of tRNA motifs by maturation enzymes. Residues that interact with tRNA are shown as stick models and are colored by subunit (green, subunit 1; blue, subunit 2). (a) Two catalytic units of the dimeric splicing endonuclease coordinately interact with the two splice junctions; the site stabilized by the green subunit is cleaved by the blue subunit and vice versa. (b) The tRNAthr precursor is primarily bound by the green subunit of tRNase Z while it is cleaved by the blue subunit. (c) Two subunits of TadA recognize the anticodon stem and loop.
the first base pair (1 and 72) and discriminator base 73 (see Figures 2b and 3b). U73 of tRNAThr unstacks from the acceptor stem to enter the active site of the enzyme, where it is co-stabilized by both subunits (see Figure 4b). www.sciencedirect.com
Complexes of tRNA and maturation enzymes Li 297
Figure 4
A tyrosine residue from one subunit and a phenylalanine residue from the other sandwich the U73 nucleobase (see Figure 3b). Unfortunately, no structural information is available for the 30 extension nucleotides, which prevents detailed analysis of the catalytic mechanism. Based on the composite RNA-binding site and the apparent flexibility of the 30 end of tRNA, however, it has been proposed that subtle differences in the dimeric arrangement and accessibility of the active site could account for the different effects of CCA on the enzymes [23]. These differences cannot explain why some enzymes can cleave both CCA-less and CCA-plus substrates, however.
Anticodon recognition by deaminase TadA and thiouridylase MnmA Anticodons play an indispensable role in codon recognition during protein translation. The structures of the anticodon of tRNA both when free [5] and when bound to ribosomes [7] reflect their ability to directly read the cognate codons in mRNA. Numerous chemical modifications are found in this region of many tRNAs after intron removal and 50 /30 trimming [33]. Recently published examples of two anticodon modifying enzymes discussed below illustrate a general mechanism for how enzymes access anticodon loop nucleotides. Both of these enzymes recognize anticodon-specific features, especially nucleotide 34, by flipping out nucleobases. TadA is a specific adenosine-to-inosine editing enzyme for wobble position 34 of bacterial tRNAArg2 [34]. This modification enables tRNA to recognize CGA/U/C codons. TadA is relatively small and functions as a homodimer [34]. In the crystal structure of Staphylococcus aureus TadA with anticodon stem-loop RNA [35], two RNA anticodon loops are bound symmetrically to the TadA dimer, in which each RNA loop is contacted by both subunits of the dimer (see Figure 2c). In particular, the dimeric enzyme coordinately recognizes the non-Watson–Crick base pair between nucleotides 32 and 38 (C32–A38) that caps the anticodon loop by forming a network of hydrogen bonds with both subunits (see Figure 3c). A prominent role for this base pair in stabilizing the anticodon hairpin and in conferring tRNA specificity has been gradually recognized [36,37]. The cross-subunit stabilization of the anticodon loop by the S. aureus TadA homodimer agrees well with the fact that yeast tRNA is edited by the heterodimeric enzyme Tad2p–Tad3p, with each of the subunits being essential [38]. Base splaying is a common feature of tRNA–maturation enzyme complexes. (a) The splicing endonuclease flips all three bulge nucleotides in the BHB motif. (b) tRNase Z flips nucleotide 73 immediately preceding the target phosphodiester bond. (c) TadA and (d) MnmA flip target nucleotide 34 for complete access to the catalytic target.
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A remarkable feature observed in the TadA–tRNAArg2 complex is the significant remodeling of anticodon nucleotides. The nucleobases of 33, 34, 35 and 37 are splayed outward to interact with the active site pocket of the enzyme (see Figure 4c). Both hydrogen-bonding and cation–p stacking interactions are used by the enzyme to stabilize the splayed bases. G36 is the only anticodon Current Opinion in Structural Biology 2007, 17:293–301
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Figure 5
Exploration of the structural flexibility of tRNA by maturation enzymes for catalytic power. In each panel, the right side shows the reaction catalyzed by the enzyme and the left side displays detailed structures at the enzyme active sites. (a) The splicing endonuclease catalyzes an intramolecular phosphotransfer reaction via a pentacordinated intermediate in an in-line conformation. The two nucleotides flanking the scissile phosphate form the in-line conformation at the enzyme active site in preparation for bond breakage. Note that the adjacent 20 -OH group was modeled into the crystal structure lacking this group. (b) Anticodon nucleotide A34 (Neb34 in the structure) is well placed to interact with a catalytic water, the catalytic zinc and the essential glutamate residue, Glu55, of TadA. (c) The intermediate of adenylated uridine in the active site of MnmA is placed for catalysis. Cys199 is suggested to be the sulfur acceptor from a sulfur relay system that is eventually transferred to the C2 atom of U34.
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Complexes of tRNA and maturation enzymes Li 299
nucleotide that points to the interior of the anticodon loop, although its nucleobase is also stabilized by hydrogen bonds with the enzyme. The catalytic target nucleotide, Neb34 (nebularine, a nucleobase analog of adenosine, was used at position 34 for co-crystallization), is the most buried nucleotide and forms several interactions with the catalytic groups (see Figure 5b). However, the lack of a 6-amino group of Neb34 is thought to prevent the enzyme from forming the prehydrolytic deamination state [35]. The network of interactions formed by the splayed anticodon nucleotides with the rigid enzyme active site and water molecules is theorized to contribute to the strength and specificity of the TadA–RNA interaction [35]. MnmA is a bacterial thiouridylase that also modifies anticodon nucleotide 34 [39]. MnmA is responsible for the conversion of U34 to s4U34 in tRNAGlu, tRNALys and tRNAGln, all of which contain UUN anticodon sequences [1]. MnmA uses an elaborate two-step mechanism to sulfurate U34 via an adenylated U34 intermediate [39]. Therefore, MnmA must recognize and fully access the unique tandem uridine nucleotides in the anticodon. MnmA binds to tRNA as a monomer, but with outspread protein domains (see Figure 2d). The protein–RNA interface extends from the anticodon loop to the bottom of the D-stem. The central domain, a glycine-rich loop in particular, reaches up to the D-stem and facilitates minor groove recognition. The catalytic domain and an EF-Tu-like domain nestle in the anticodon region, in which the two uridine nucleotides are most extensively stabilized by hydrogen bonds. This stabilization is, once again, facilitated by extruded bases. The catalytic target uridine is completely extruded from the stacked anticodon into the deep pocket of the catalytic domain (see Figures 2d and 4d). The other two anticodon nucleotides, however, remain partially stacked in the enzyme (see Figures 2d and 4d). The resulting active site, especially after adenylation of U34, forms a closed chamber in preparation for the sulfur-transfer reaction (see Figure 5c).
Conclusions Two overriding themes evident from these structural studies expand characteristics uncovered in other tRNA complex studies: significant flexibility of RNA at the processing or modifying sites, and composite (or crosssubunit) RNA recognition by multiple enzyme subunits. Structural remodeling seems to be the most direct method for enzymes to access folded RNA bases. Besides those described here and aminoacyl-tRNA synthetases, other tRNA maturation enzymes also adopt this strategy to access their catalytic targets (Table 1). These include class I and II tRNA CCA-adding enzymes [40,41,42], amidation transferase GatDE [43], G15 and G34 transglycosylases [44–46] and U55 pseudouridylase [46–48]. Together, these examples suggest that many modifying www.sciencedirect.com
and processing enzymes exploit the intrinsic flexibility of tRNA to gain catalytic power towards specific targets. In addition, the structural flexibility of RNA provides a means for some enzymes to achieve organism-specific RNA recognition. Whereas relatively rigid active sites are intolerant of slight variations in the target bases of RNA substrates, more elastic active sites of the homologous enzymes may permit the variations by ‘scrunching’ the bases. An extraordinary example of the scrunching model was elegantly demonstrated for the class I CCA-adding enzyme. The three nucleotides at the 30 end of a tRNA are added in a nucleotide-wise fashion by the single active site of the CCA-adding enzyme [41,42]. It remains to be seen whether the patterns of substrate specificity for splicing endonucleases and tRNase Z can be explained by similar models. Subunit composition is another facet to the mechanism of tRNA maturation enzymes. Regardless of the number of catalytic target sites, the enzymes discussed here have multiple subunits or domains that form composite binding sites for RNA substrates. By jointly recognizing RNA features, composite binding ensures substrate specificity. In general, oligomerization enhances binding affinity by increasing the amount of surface that contacts the substrate and can regulate enzyme activity according to the intracellular enzyme concentration. Furthermore, oligomerization affords the flexibility for individual subunits to specialize, potentially enabling functional connections with other cellular machines. The evolutionary histories of the enzymes discussed here have already begun to shed light on this hypothesis [49].
Looking into the future tRNA maturation enzymes catalyze some of the most elaborate reactions known, which makes catalytic strategy a fascinating counterpart to the study of tRNA recognition. What are the functional groups in tRNA and enzymes that stabilize reaction intermediates, and how are these related to the intrinsic flexibility of RNA and enzyme organization? The excitement about elucidating these mechanisms is now well placed.
Acknowledgements The author is grateful for scholarly discussions with Eric Westhof, Anita Marchfelder, Dieter So¨ll, Robert Reeves, Christopher Trotta, Salvatore San Paolo, Michaeal P Terns, Rebecca M Terns, Wei Yang, Kate Calvin and Song Xue. The author thanks the National Institutes of Health (R01 GM66958-01) and National Science Foundation (MCB-0517300) for financial support.
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Saenger W: Principles of Nucleic Acid Structure. New York: Springer-Verlag; 1984.
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18. Arnez JG, Moras D: Structural and functional considerations of the aminoacylation reaction. Trends Biochem Sci 1997, 22:211-216. 19. Nakanishi K, Nureki O: Recent progress of structural biology of tRNA processing and modification. Mol Cells 2005, 19:157-166. This review summarizes earlier structural studies on tRNA processing and modification enzymes. The described enzyme structures include RNase PH, tRNase Z, CCA-adding enzymes, archaeosine tRNA guanine-transglycosidases, queuosine tRNA guanine transglycosidase, U55 pseudouridine synthase TruB and RNA methyltransferase TrmH. Current Opinion in Structural Biology 2007, 17:293–301
34. Wolf J, Gerber AP, Keller W: TadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J 2002, 21:3841-3851. 35. Losey HC, Ruthenburg AJ, Verdine GL: Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA. Nat Struct Mol Biol 2006, 13:153-159. The tRNA adenosine deaminase TadA is an essential bacterial enzyme responsible for adenosine-to-inosine editing of the wobble base (A34) of tRNAArg2. The edited tRNA is capable of reading CUA/U/C codons. The co-crystal structure of S. aureus TadA bound to the anticodon stem-loop of tRNAArg2 bearing nebularine, a non-hydrolyzable adenosine analog, www.sciencedirect.com
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shows a network of interactions between the enzyme and the RNA. Anticodon nucleotides are splayed in the complex to facilitate the specificity of recognition and catalysis. This recognition is facilitated by dimerized enzyme subunits and explains the specificity of the enzyme for tRNAArg2. 36. Auffinger P, Westhof E: Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes. J Mol Biol 1999, 292:467-483. 37. Olejniczak M, Uhlenbeck OC: tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition. Biochimie 2006, 88:943-950. 38. Gerber AP, Keller W: An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science 1999, 286:1146-1149. 39. Numata T, Ikeuchi Y, Fukai S, Suzuki T, Nureki O: Snapshots of tRNA sulphuration via an adenylated intermediate. Nature 2006, 442:419-424. 20 -thio modification of uridine at the first anticodon position (U34) of tRNAGlu, tRNALys and tRNAGln is phylogenetically conserved. This is carried out by MnmA thiouridylase via two enzymatic steps and an adenylated nucleotide intermediate. The co-crystal structures of E. coli MnmA and tRNAGlu in three functional states illustrate one principle of tRNA anticodon stem-loop recognition, similar to what is observed for TadA; anticodon nucleotide U34 is splayed outwards to reach the enzyme active site for modification. Active site rearrangement enables identification of catalytically important residues. Two possible catalytic mechanisms are proposed. 40. Xiong Y, Steitz TA: A story with a good ending: tRNA 30 -end maturation by CCA-adding enzymes. Curr Opin Struct Biol 2006, 16:12-17. 41. Xiong Y, Steitz TA: Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 2004, 430:640-645. The CCA-adding enzyme is responsible for the addition of the 30 -terminal CCA sequence to tRNA. Crystal structures of the Archaeoglobus fulgidus tRNA nucleotidyltransferase (a class I CCA-adding enzyme) in complex with yeast tRNAPhe at low resolution and in three distinct complexes with the acceptor stem at better than 3.4 A˚ resolution offer significant insights into how CCA is polymerized without a nucleic acid template. The remarkable series of images captured at each enzymatic step — after the addition of C74, after the addition of C75 and after the addition of A76 — showed how the enzyme can switch specificity from CTP to ATP and that the enzyme active site is progressively altered by the elongating 30 end of the tRNA. However, the lack of the TCC loop in the model acceptor stem, which contacts the RNA-binding tail domain, could affect the precise ways in which the RNA and the active site residues rearrange in these crystals. 42. Tomita K, Ishitani R, Fukai S, Nureki O: Complete crystallographic analysis of the dynamics of CCA sequence addition. Nature 2006, 443:956-960.
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This work describes six distinct complexes of the A. fulgidus CCA-adding enzyme with a TCC-loop-containing minihelix derived from Thermotoga maritima tRNAPhe, and with or without incoming nucleotides. Significant protein and RNA dynamics were observed at different polymerizing stages; however, these were somewhat different from those described in [41]. The acceptor stem expands and contracts in the first polymerization step. The subsequent steps are similar to those described in [41] in that the primer nucleotides splay out of the way so that the incoming NTP can be accommodated in the catalytic cleft. The sidechain of an important arginine residue was observed in different conformations depending on the polymerization stage; this is believed to achieve CTP/ATP discrimination. 43. Oshikane H, Sheppard K, Fukai S, Nakamura Y, Ishitani R, Numata T, Sherrer RL, Feng L, Schmitt E, Panvert M et al.: Structural basis of RNA-dependent recruitment of glutamine to the genetic code. Science 2006, 312:1950-1954. The amidotransferase GatDE is a heterodimeric enzyme that converts the misacylated Glu-tRNAGln by amidation to Glu-tRNAGlu as a unique charging process in archaea. GatDE uses separate subunits to produce ammonia, to phosphorylate the g-carboxyl group of the tRNA-bound glutamate and to convert phosphoglutamate to glutamine. This work describes a co-crystal structure of Methanothermobacter thermautotrophicus GatDE in complex with tRNAGln at 3.2 A˚ resolution. The structure reveals a surprising method of tRNAGln recognition that is independent of anticodon. The shape and the tRNAGln-specific bases of the acceptor stem are recognized by the enzyme. The structure also sheds light on the mechanism of ammonia recruitment through an 40 A˚ tunnel and repeating hydrogen bonding. 44. Xie W, Liu X, Huang RH: Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat Struct Biol 2003, 10:781-788. 45. Ishitani R, Nureki O, Nameki N, Okada N, Nishimura S, Yokoyama S: Alternative tertiary structure of tRNA for recognition by a posttranscriptional modification enzyme. Cell 2003, 113:383-394. 46. Ferre-D’Amare AR: RNA-modifying enzymes. Curr Opin Struct Biol 2003, 13:49-55. 47. Hoang C, Ferre-D’Amare AR: Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme. Cell 2001, 107:929-939. 48. Pan H, Agarwalla S, Moustakas DT, Finer-Moore J, Stroud RM: Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit. Proc Natl Acad Sci USA 2003, 100:12648-12653. 49. Paushkin SV, Patel M, Furia BS, Peltz SW, Trotta CR: Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 30 end formation. Cell 2004, 117:311-321.
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