RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon
2009; Springer Nature; Volume: 28; Issue: 9 Linguagem: Inglês
10.1038/emboj.2009.69
ISSN1460-2075
AutoresSarin Chimnaronk, Tateki Suzuki, Tetsuhiro Manita, Yoshiho Ikeuchi, Min Yao, Tsutomu Suzuki, Isao Tanaka,
Tópico(s)RNA Research and Splicing
ResumoArticle26 March 2009free access RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon Sarin Chimnaronk Sarin Chimnaronk Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, Thailand Search for more papers by this author Tateki Suzuki Tateki Suzuki Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Tetsuhiro Manita Tetsuhiro Manita Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Yoshiho Ikeuchi Yoshiho Ikeuchi Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Min Yao Min Yao Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Tsutomu Suzuki Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Isao Tanaka Corresponding Author Isao Tanaka Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Sarin Chimnaronk Sarin Chimnaronk Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, Thailand Search for more papers by this author Tateki Suzuki Tateki Suzuki Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Tetsuhiro Manita Tetsuhiro Manita Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Yoshiho Ikeuchi Yoshiho Ikeuchi Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Min Yao Min Yao Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Tsutomu Suzuki Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Isao Tanaka Corresponding Author Isao Tanaka Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Author Information Sarin Chimnaronk1,2, Tateki Suzuki1, Tetsuhiro Manita1, Yoshiho Ikeuchi3, Min Yao1, Tsutomu Suzuki3 and Isao Tanaka 1 1Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan 2Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, Thailand 3Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan *Corresponding author. Faculty of Advanced Life Sciences, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan. Tel.: +81 11 706 3221; Fax: +81 11 706 4905; E-mail: [email protected] The EMBO Journal (2009)28:1362-1373https://doi.org/10.1038/emboj.2009.69 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Post-transcriptional RNA modifications in the anticodon of transfer RNAs frequently contribute to the high fidelity of protein synthesis. In eubacteria, two genome-encoded transfer RNA (tRNA) species bear the same CAU sequence as the anticodons, which are differentiated by modified cytidines at the wobble positions. The elongator tRNAMet accepts an acetyl moiety at the wobble base to form N4-acetylcytidine (ac4C): an inherent modification ensures precise decoding of the AUG codon by strengthening C−G base-pair interaction and concurrently preventing misreading of the near cognate AUA codon. We have determined the crystal structure of tRNAMet cytidine acetyltransferase (TmcA) from Escherichia coli complexed with two natural ligands, acetyl-CoA and ADP, at 2.35 Å resolution. The structure unexpectedly reveals an idiosyncratic RNA helicase module fused with a GCN5-related N-acetyltransferase (GNAT) fold, which intimately cross-interact. Taken together with the biochemical evidence, we further unravelled the function of acetyl-CoA as an enzyme-activating switch, and propose that an RNA helicase motor driven by ATP hydrolysis is used to deliver the wobble base to the active centre of the GNAT domain. Introduction Most cellular RNAs require post-transcriptional chemical modifications to be mature and functional (McCloskey and Crain, 1998). Among more than a hundred modified ribonucleosides identified to date, the vast majority are found in transfer RNA (tRNA), particularly embedded in the anticodon loop region (Grosjean et al, 1996). Modified bases at the first (wobble) position of the tRNA anticodons are involved in maintaining the fidelity of genetic information transfer by stabilizing specific codon–anticodon interactions to prevent misreading of non-cognate codons, and to ensure the precise reading frame (Björk et al, 1999; Suzuki, 2005; Agris et al, 2007). Additionally, wobble modifications coincidentally serve as determinants for recognition by cognate aminoacyl-tRNA synthetases (aaRSs) (Muramatsu et al, 1988a; Sylvers et al, 1993; Senger et al, 1997). In γ-proteobacterial cells including Escherichia coli, the decoding system for AUR (R=A or G) codons is dominated by a pair of the modifications at the wobble positions of the two tRNAs that share the same CAU anticodons: the AUA codon-specific isoleucine tRNA (tRNAIle2) and the elongator tRNAMet responsible for AUG decoding (Supplementary Figure S1). Modifying the wobble cytidine base of tRNAIle2 to lysidine (k2C) results in a conversion of codon and amino-acid specificities from AUG to AUA and methionine to isoleucine, respectively (Muramatsu et al, 1988a, 1988b). The essential enzyme that synthesizes lysidine at the first letter of the anticodon of tRNAIle2 was previously identified as TilS (tRNAIle-lysidine synthetase) and characterized (Soma et al, 2003; Ikeuchi et al, 2005). Meanwhile, the elongator tRNAsMet contain specific N4-acetylcytidine (ac4C) at the same position. This modified wobble base has been reported to stabilize the C3′-endo puckering conformation of ribose, encouraging the C−G base-pair interaction, thereby ensuring the decoding of the AUG codon as methionine (Kawai et al, 1989). Moreover, in vitro translation experiments using E. coli tRNAMet suggested that ac4C also has an important function in preventing errors that may arise during protein synthesis due to misreading of isoleucine AUA codon by tRNAMet (Stern and Schulman, 1978). Hence, these two modification enzymes that generate a pair of wobble modifications essentially determine the accuracy of the decoding property of each tRNA, and the protein synthesis as well. Most recently, Suzuki and colleagues (Ikeuchi et al, 2008) have successfully identified a non-lethal gene, ypfI, responsible for ac4C formation at the wobble base of the elongator tRNAMet, which therefore has been renamed tmcA (tRNAMet cytidine acetyltransferase). E. coli tmcA, which belongs to the orthologous cluster COG1444, encodes an uncharacterized enzyme of 671 amino-acid residues with a molecular mass of 75 kDa. Cautious inspection of the sequence alignments revealed a pair of highly conserved regions: the DUF699 and Acetyltransf_1 Pfam domains near the amino and carboxyl termini, respectively. DUF699 is approximately 150 amino-acid residues long and appears to have a distinctive ‘AxRGRGKS’ P-loop (also known as Walker A) motif, implying inherent ATPase activity. Indeed, in vitro synthesis of ac4C revealed that TmcA exploits acetyl-CoA as an acetyl group donor, which is transferred in an ATP-dependent manner (Ikeuchi et al, 2008). Neither structural information nor likely roles of DUF699 ATPase activity in ac4C synthesis have so far been elucidated. In this study, to provide the precise mechanistic insight into site-specific RNA acetylation that maintains the decoding fidelity in bacterial translation system, we solved the crystal structure of E. coli TmcA bound with acetyl-CoA and ADP at a resolution of 2.35 Å. In the structure, TmcA reveals a unique four-domain configuration in which acetyl-CoA- and nucleotide-binding sites are unexpectedly separated by 30 Å. Strikingly, with no detectable sequence similarity, the peripheral N-terminal and DUF699 domains structurally mimic the DEAD-box RNA helicases, forming duplicated RecA-like fold in a non-canonical inverted topology as the P-loop resides in the C-terminal part. Combining our structural interpretation with a wealth of biochemical studies, we propose an unprecedented model for an RNA-modification enzyme that incorporates an RNA helicase module designed to remodel the substrate structure, and give access to the modified sites. Results Overall structure of E. coli TmcA complexed with Acetyl-CoA and ADP Our initial attempts to crystallize the full-length E. coli TmcA fused with a hexahistidine tag at either the N or C terminus were unsuccessful. Both tag removal and supplementation with acetyl-CoA were indispensable for yielding quality crystals (Supplementary Figure S2). Finally, the structure of TmcA was solved in the orthorhombic space group P212121 at 2.35-Å resolution using a selenomethionine-substituted protein, and refined to an Rfree of 0.274 and R-factor of 0.234 (Table I). The two TmcA molecules in an asymmetric unit (labelled A and B to distinguish them) were essentially identical, with a root mean square deviation (r.m.s.d.) of 0.9 Å for all corresponding Cα atoms. ATP and MgCl2 were also included in the crystallization conditions with the aim of characterizing the ATP-binding and hydrolysis activities of TmcA. Unexpectedly, there was only one detectable ADP molecule, presumably resulting from the TmcA-catalysed ATP hydrolysis, bound to monomer A. In contrast, the site that was occupied by the β-phosphate in monomer A bore obvious density for a sulphate ion in monomer B (Supplementary Figure S2). The absence of a nucleotide cofactor in monomer B likely gave rise to higher temperature B-factors in the DUF699 domain, and thus we hereafter focus on monomer A for structural description, unless otherwise stated. Table 1. Summary of crystallographic data and refinement statistics Data collection Selenium crystal 1 Selenium crystal 2 Data set Peak Edge Remote Space group P212121 P212121 Unit cell (Å) a=61.1, b=101.2, c=263.5 a=61.3, b=101.0, c=263.1 Wavelength (Å) 0.9791 0.9797 0.9645 1.0000 Resolution (Å)a 50–2.90 (3.00–2.90) 50–2.90 (3.00–2.90) 50–2.90 (3.00–2.90) 50–2.35 (2.43–2.35) Observed total/unique reflections 266 351/36 980 268 698/36 912 265 695/36 883 425 934/66 310 Completeness (%)a 100.0 (100.0) 100.0 (100.0) 100.0 (100.0) 96.6 (92.1) Rmerge (%)a,b, a,b 13.5 (64.0) 13.2 (65.5) 13.6 (67.0) 5.6 (37.3) I/σ (I)a 9.1 (2.4) 9.0 (2.4) 8.9 (2.3) 25.3 (4.7) Redundancya 7.2 (7.3) 7.3 (7.4) 7.2 (7.3) 6.4 (6.1) Refinement Resolution (Å) 20–2.35 Number of reflections (working/free) 61 032/5079 R-factor/Rfree (%)c 23.4/27.4 Protein/ligand/ion/water atoms 10 411/129/5/350 Ramachandran (%)d 88.5/11.3/0.0/0.2 r.m.s.d. bonds (Å)/angles (deg) 0.015/1.99 Average B-value (Å2) 47.5 a Values in the parentheses refer to the highest resolution shell. Rmerge=∑j∑i∣〈Ij〉−Ij,i∣/∑j∑iIj,i, where 〈Ij〉 is the average intensity of reflection j for its symmetry equivalents. c R-factor=∑∥Fobs∣−∣Fcalc∥/∑∣Fobs∣. Rfree is calculated for a randomly chosen 7% of total reflections that were not used for structure refinement. d Fractions of residues in the most favoured/allowed/generously allowed/disallowed regions of the Ramachandran plot. Overall, the current structure is well ordered except for 11 residues (55−65) in the central portion of the extended loop flanked by the β2- and β3-strands, which were not modelled. TmcA is apparently a four-domain protein in an L-shaped morphology with the dimensions of 60 Å × 70 Å × 70 Å, composed of three mixed α/β-domains and a single exclusively helical domain at the C terminus (Figure 1B). TmcA represents a novel structure with unique domain organization based on a search for similar structures by the DALI engine. The peripheral N-terminal globular domain (hereafter referred to as ‘the head domain’) is formed by the N-terminal residues 1–171 and residues 321–336 from the middle, constructing a central parallel five-stranded β-sheet (β1–β5) sandwiched by α-helices on both sides (Figure 1B and F). The juxtaposed α/β-domain is the Pfam-classified DUF699 domain (residues 172–320), which is connected to the head domain by a lengthy linker (∼60 Å) that folds back onto the C terminus of the α1 helix. Apparently, the size and conformation of the DUF699 domain closely resembles those of the head domain with a few additions of a β-strand and two flanking short helices on one side. Superposition of the head and DUF699 domains of TmcA gives a moderate r.m.s.d. of 3.4 Å for 108 pairs of Cα atoms compared. This observation was hardly predictable from sequence comparison as DUF699 and the head domains share only 12% amino-acid sequence identity. Figure 1.Overall structure of E. coli TmcA. (A) Sequence alignment of the seven consensus motifs found in TmcA and its homologues from all kingdoms. E. coli TmcA used for structure determination is in the top row and is numbered. Seven other homologues from Haemophilus influenzae, Vibrio vulnificus, Pyrococcus horikoshii, Haloarcula marismortui, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens are listed from the top. (B) Ribbon representation of the TmcA crystal structure shown in a top–down view. Each domain is differently coloured and labelled. Bound ADP and acetyl-CoA are depicted in a ball-and-stick representation. (C) An edge-on view reveals an L-shaped pot with an acetyl-CoA plug. (D) Solvent-accessible surface is shown in the same orientation as (B) and is coloured according to the electrostatic potential calculated by the program GRASP (blue for positively charged and red for negatively charged). Bound ADP and acetyl-CoA are drawn as spherical models. (E) Coordination of ADP by the DUF699 domain. Side chains involved in interactions with ADP are depicted in a ball-and-stick representation and are labelled. For clarity of the hydrogen-bonded network (cyan dots), the backbones of the motif I (P loop) are drawn as sticks. Water molecules are shown as cyan spheres. Nitrogen atoms are coloured in blue, and oxygen in red. The omit Fo−Fc difference electron density map (3.5 σ, magenta) of ADP is superimposed onto the refined coordinates. (F) Topology diagram of TmcA. The domains are coloured as in (B) and secondary elements are labelled. Download figure Download PowerPoint The C-terminal tail of DUF699 domain folds back again into the head domain and sticks an α12 helix on the backside of the β-sheet in the head domain (Figure 1F). A short helix (η3) following α12 connects two N-terminal repeated α/β-domains with the middle catalytic ‘body’ domain. The body domain of TmcA is made by a planar mixed α/β-topology containing a central seven-stranded β-sheet (β12–β18) bordered by six α-helices (α13–α17, and α20) and backed by two α-helices (α18 and α19). It possesses an Acetyltransf_1 Pfam signature, indicating the acetyltransferase catalytic centre. Indeed, acetyl-CoA binds to a wedge-like opening in the centre of the domain, which is made by breaking the two parallel β15 and β16 strands by halves. A characteristic ‘β-bulge’ that contributes to this distortion is formed by a bifurcated hydrogen bond between the main chains of Trp410 and the two adjacent Ser459–Arg460 residues. At the end of the body, the bordering α20 helix makes hydrogen-bonding interactions with the capping ‘tail domain’, and is kinked at Arg550 triggered by a salt bridge with Asp577 on the α21 helix in the tail domain. The tail domain is an antiparallel bundle of six helices with alternating up–down topology (α21–α26), forming a bulky helical bundle together with α16, α17, and η4 of the body domain. This creates a shallow positively charged inner concave by clusters of arginine and histidine residues, which plausibly contribute to the tRNA-binding activity (Figure 1D). The N-terminal head and DUF699 domains structurally mimic DEAD-box helicases The two tandemly repeated α/β-domains at the N terminus of TmcA seemingly reveal structural duplication of a module within the molecule with a great deal of amino-acid residue substitution. They are oriented in such a way that the β-sheets lie approximately 60° to each other, and are in close proximity, so that they can be arranged to form a deep interdomain cleft where ADP binds. The presumable phosphate-binding P-loop (Walker A) motif resides in the DUF699 domain flanked by α7 and β6, and is absent in the head domain. This appearance is reminiscent of the DEAD-box RNA helicases that are composed of two successive RecA-like domains. As expected, the highest structural similarity to the head and DUF699 domains was found in a DEAD-box protein from the hyperthermophile Methanococcus jannaschii (1HV8; Story et al, 2001), with a Cα r.m.s.d. of 3.1 Å (Figure 2A). The relative orientation of the head and DUF699 domains in the TmcA structure differs significantly from that in the ‘closed’ form of the DEAD-box protein complexed with poly(U) RNA and non-hydrolysable AMPPNP (Kim et al, 1998; Sengoku et al, 2006). The structure most likely represents an alternative ‘open’ state prior to accommodating RNA substrate as observed in other core structures of DEAD-box proteins, such as eIF4A, MjDEAD, and UAP56 (Caruthers et al, 2000; Story et al, 2001; Shi et al, 2004). Bearing this in mind, binding of tRNAMet to TmcA possibly causes a drastic movement of the DUF699 domain towards the head and body domains, which may be necessary for the recognition and/or the positioning of tRNA. Figure 2.Structural comparison to well-known RNA helicases. (A) Superposition of the DUF699 domain of TmcA onto the N-terminal RecA-like domains of various DEAD-box RNA helicases. The C-terminal domains of both Vasa and HCV NS3 helicases rotate towards the N-terminal domains revealing a closed conformation upon binding of the substrate RNAs (both 5′ and 3′ termini are marked). (B) In the upper panel, the universally conserved sequence motifs among the DEAD-box helicases are mapped onto the Vasa structure. Each motif is differently coloured and amino-acid sequences are given. Symbols used are as follows. x: any amino-acid residue; o: small hydrophilic residues such as serine or threonine; h: hydrophobic residues. Bound poly(U) RNA is illustrated as dark sticks. Mg2+ is shown as a green sphere. The canonical helicase motifs are structurally superimposed onto the conserved sequences in the N-terminal region of TmcA (lower panel). Note that the two N-terminal domains of TmcA are in an inverted topology compared with Vasa (i.e. the P-loop resides in the N-terminal domain of Vasa but it resides in the C-terminal domain of TmcA). Download figure Download PowerPoint To date, all characterized DEAD-box proteins carry a core composed of two RecA-like domains with the P loop always occurring in the N-terminal domain. Remarkably, in contrast to the universal topology of the DEAD-box helicases, TmcA harbours the ATPase site in the DUF699 domain C-terminal to the head domain, that is, in a permuted topology (Figure 2B). This is ascribed to a stretched turn loop between β5 and α6, by which the head and DUF699 domains are linked. This dramatic topological difference between TmcA and the DEAD-box helicases explains why earlier sequence analyses could not envisage their structural resemblance. A number of well-known conserved motifs in the DEAD-box helicases are lined along the interdomain interfaces between the two RecA-like domains (Figure 2B) (Tanner and Linder, 2001; Cordin et al, 2006), where mutual inter-motif interactions have an important function in coupling ATP hydrolysis to helicase activity (Sengoku et al, 2006). Although the sequences in these regions are hardly preserved in the N-terminal domains of TmcA, most elements can be annotated in terms of an equivalent structural relationship. It is noted that these pseudo-helicase motifs are also strictly conserved in TmcA among species (Supplementary Figure S3). Accordingly, we readily noticed a motif II (Walker B motif)-like sequence at the end of the β9 strand with a remarkable DEAA sequence. Furthermore, we discovered three unrecognized sequence motifs that we designated TmcA-specific (TS) motifs 1, 2, and 3, following the aberrant DEAA motif in TmcA (Figure 2B; Supplementary Figure S3). On the basis of the structural relationship, the TS1 (YEGxG) and 2 (hRW/Y) motifs correspond to motifs III and VI of the DEAD-box proteins, respectively. The invariant Ser-Ala-Thr (SAT) sequence in motif III, which is essential for RNA-unwinding activity but not ATPase activity for DEAD-box helicases (Pause and Sonenberg, 1992), is no longer present in the TS1 motif. The large-scale amino-acid substitution in the corresponding helicase motifs of TmcA could be rationalized by intimate interactions among motifs II, III, and VI (DEAD, SAT, and HRIGR) seen in the complex structures of DEAD-box helicases with bound synthetic poly(U) RNA (Bono et al, 2006; Sengoku et al, 2006). Long-range communication between the two remote active sites The nucleotide-binding pocket is formed within a cleft between the head and the DUF699 domains. TmcA has intrinsic hydrolysis activity as implied in the crystal structure, and described in recently published paper (Ikeuchi et al, 2008). The adenine base is held in a hydrophobic pocket between Phe236 and Ile318 (Figure 1E). Ile318 is a conserved hydrophobic side chain in the TS2 motif, and also makes a van der Waals interaction with the ribose ring, together with Val232 and the methylene group of Arg319. The adenine base is specifically selected through bifurcated hydrogen bonds with Gln180 in the Q motif, and further assured through hydrogen bonding with Gln211. From the TS2 motif, Arg319 contacts the ribose 3′-OH. TmcA may not be able to rigorously differentiate dATP or GTP from ATP as the 2′-OH is not discerned and the interacting glutamine side chain is capable of acting both as the hydrogen acceptor and the donor against the C6 exocyclic groups of adenine and guanine, respectively. As predicted, these were possible substrates for the hydrolysis reaction (Ikeuchi et al, 2008). The more promiscuous use of NTPs has so far been reported in cases of DEAH-box proteins (for example, see Tanaka and Schwer, 2006). On the other hand, the phosphate moiety is directly recognized by the invariant Lys205 and Ser206 residues in the P loop, and is anchored by the backbone amide nitrogens of residues 202–207 in the P loop. ADP binds to the hydrolysis pocket of TmcA in a way compatible with that of Vasa except for a rotation of the adenine base by 48° around the N-glycosidic bond. It is notable that the second arginine residue in motif VI (HRxGRxGR) of DEAD-box proteins, which has been proposed to function as an ‘arginine finger’ stabilizing the transition state of the hydrolysed intermediate (Scheffzek et al, 1997; Caruthers and McKay, 2002), is replaced by an extra Arg201, the first arginine in the P loop (AxRGRGKS) of TmcA. Around 30 Å away from the hydrolysis site is an acetyl-CoA-bound slot, where the transfer of the acetyl group to RNA should be carried out. The region encompassing the cofactor-binding site, ‘the body domain’, revealed the structure most homologous to the ubiquitous GCN5-related N-acetyltransferase (GNAT) superfamily, which comprises the nuclear histone acetyltransferases (HATs) and others (Supplementary Figure S4) (Vetting et al, 2005). The two sequence motifs of the GNAT folds, motifs GNAT A and GNAT B, form the essence of the acetyl-CoA-binding site in TmcA. The cofactor acetyl-CoA is trapped through direct hydrogen bonds between its pyrophosphate group and main chain amides of residues Arg469–Arg474 at the amino end of α18 in the GNAT A motif, and between its 3′-phosphate group and Arg506 in the GNAT B motif (Supplementary Figure S4). The pantetheine group lies on the hydrophobic platform made by both β15 and β16 in the GNAT motifs A and B, respectively, and is sharply bent, probably due to a specific interaction with Gln468. The adenine ring is recognized by Glu499 in motif B, whereas the acetyl group on the other end is barely constrained. The nearest atom to the carbonyl oxygen of acetyl-CoA is the backbone amide nitrogen of Ile461, which is 3.7 Å away. In the RNA modification process, ATP is often utilized by enzymes for the activation of premodified sites through an adenylate intermediate prior to nucleophilic substitution by the donors. Recently solved structures of tRNA-modifying enzymes (e.g. TilS and MnmA) hold both ATPs and substrates within the immediate vicinity to prevent undesirable hydrolysis of the labile intermediate (Numata et al, 2006). As acetyl-CoA is an activated carrier, it is intuitive that TmcA should not require ATP for RNA base activation to synthesize ac4C. However, in vitro analysis of TmcA demonstrated that ATP hydrolysis was essential for ac4C formation (Ikeuchi et al, 2008). In addition, we were aware of a channel that is flanked on both sides by a line of the most characteristic conserved motifs, which orchestrates an extensive and complicated interaction network combining two remote active sites (Figure 4A). From the ATPase centre, Arg203 in the middle of the P loop is in a van der Waals contact with Pro317, and forms a salt bridge with Glu327 in the TS2 motif, which in turn is hydrogen-bonded to Trp320. The P loop also directly interacts with the TS1 motif through a putative arginine finger (Arg201) sandwiched by Tyr291 and Thr287, and is further bridged indirectly to the TS3 motif by electrostatic interactions formed along the path Arg201 (I)−Glu292 (TS1)−Arg379 (TS3). There is a characteristic threonine cluster (residues 285, 286, 287, and 294) at the bottom of the TS1 hairpin, where the P loop, the motif II, and the TS1 motif are linked together through Ser206 (I)−Asp262 (II)−Tyr285 (TS1) with a water molecule. Finally, two residues, His 377 and Asp384, in the TS3 motif cooperatively pin Arg460, the residue juxtaposed to Ile 461 in the GNAT A motif. It is noteworthy that almost all relevant residues listed above are unique to and evolutionarily conserved in TmcA; therefore, this long-range interaction network is quite different from that of DEAD-box helicases (Sengoku et al, 2006), and may represent an unparalleled remote communication that has an important function in coupling ATPase with acetyltransferase activities in TmcA (see below). Acetyl-CoA switches on both ATPase- and tRNA-binding activities Several lines of evidence have indicated that typical RNA helicases hydrolyse ATP in an RNA-dependent manner (Cordin et al, 2006). At the same time, HATs and probably other GNAT proteins have an ordered Bi-Bi mechanism for acetyl-CoA-dependent substrate-specific binding (Lau et al, 2000; Tanner et al, 2000). What is the case with TmcA, in which both modules are integrated? To seek out the undefined roles of cofactors in tRNAMet binding, we carried out electrophoretic mobility shift assays (EMSAs) using in vitro transcribed unmodified tRNAs as substrates (Figure 3A). The results confirmed a high potential ability of TmcA to discriminate tRNAMet from tRNAIle2 (Figure 3B). However, specific binding activity was quite low in the absence of cofactors, and was not turned up by additions of either ATP or its non-hydrolysable derivative, ADPCP. In contrast, the acetyl-CoA cofactor boosted tRNA-binding activity, and was essential for the formation of a stable TmcA−tRNAMet complex. Supplying this prereaction complex with ATP, but not ADPCP, resulted in the release of tRNA from the complex as the reaction was completed. Coenzyme A alone could not retain tRNA-binding activity, nor did CoAs with bulky thioesters (Figure 3A and B). These results, therefore, underline an achievement of high selectivity for both RNA substrate and cofactor by TmcA, and suggest that the acetyl moiety has a key function in the rational ‘up- and downregulation’ of tRNA-binding affinity during the course of RNA acetylation. Figure 3.Electrophoretic mobility shift assays (EMSAs) using in vitro transcribed tRNAs. (A) EMSA with the natural substrates. Non-denaturing polyacrylamide gels were stained with Coomassie Brilliant Blue (upper panel) or ethidium bromide (lower panel) to visualize proteins and RNA, respectively. Bands corresponding to 50 pmol recombinant TmcA protein and tRNAMet are shown in the first and second lanes, respectively. Loaded protein was fixed at 50 pmol throughout the experiments, whereas the amount of tRNA ranged from 25 (× 0.5 times excess of enzyme) to 200 (× 4) pmol. The chemical structure of CoA is illustrated at the top of its lane but only the terminal moiety is shown for acetyl-CoA. Arrows indicate the positions of TmcA–tRNA complex bands. Asterisk indicates uncharacterized bands possibly responsible for the nonspecific binding of TmcA to RNA due to the extensive positively charged surface. (B) EMSA with bulky thioester groups as well as tRNAIle. Download figure Download PowerPoint Regarding hydrolysis activity, TmcA has, unlike the DE
Referência(s)