Escherichia coli tRNA(4)(Arg)(UCU) induces a constrained conformation of the crucial Omega-loop of arginyl-tRNA synthetase

Previous investigations show that tRNA(Arg)-induced conformational changes of arginyl-tRNA synthetase (ArgRS) Omega-loop region (Escherichia coli (E. coli), Ala451-Ala457) may contribute to the productive conformation of the enzyme catalytic core, and E. coli tRNA(2)(Arg)(ICG)-bound and -free conformations of the Omega-loop exchange at an intermediate rate on NMR timescale. Herein, we report that E. coli ArgRS catalyzes tRNA(2)(Arg)(ICG) and tRNA(4)(Arg)(UCU) with similar efficiencies. However, 19F NMR spectroscopy of 4-fluorotryptophan-labeled E. coli ArgRS reveals that the tRNA(4)(Arg)(UCU)-bound and -free conformations of the Omega-loop region interconvert very slowly and the lifetime of bound conformation is much longer than 0.33 ms. Therefore, tRNA(4)(Arg)(UCU) differs from tRNA(2)(Arg)(ICG) in the conformation-exchanging rate of the Omega-loop. Comparative structure model of E. coli ArgRS is presented to rationalize these 19F NMR data. Our 19F NMR and catalytic assay results suggest that the tRNA(Arg)-induced conformational changes of Omega-loop little contribute to the productive conformation of ArgRS catalytic core.     

Determinants in tRNA for activation of arginyl-tRNA synthetase: evidence that tRNA flexibility is required for the induced-fit mechanism

Arginyl-tRNA synthetase (ArgRS) catalyzes formation of arginyl-adenylate in a tRNA-dependent reaction. Previous studies have revealed that conformational changes occur upon tRNA binding. In this study, we analyzed the sequence and structural features of tRNA that are essential to activate the catalytic center of mammalian arginyl-tRNA synthetase. Here, tRNA variants with different activator potential are presented. The three regions that are crucial for activation of ArgRS are the terminal adenosine, the D-loop, and the anticodon stem-loop of tRNA. The Add-1 N-terminal domain of ArgRS, which has the very unique property among aminoacyl-tRNA synthetases to interact with the D-loop in the corner of the convex side of tRNA, has an essential role in anchoring tRNA and participating in tRNA-induced amino acid activation. The results suggest that locking the acceptor extremity, the anticodon loop, and the D-loop of tRNA on the catalytic, anticodon-binding, and Add-1 domains of ArgRS also requires some flexibility of the tRNA molecule, provided by G:U base pairs, to achieve the productive conformation of the active site of the enzyme by induced fit.     

In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase

Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.     

一个高效表达、快速纯化大肠杆菌精氨酰-tRNA合成酶的新系统(英文)

编码大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶（ Ａｒｇ Ｒ Ｓ） 的基因ａｒｇ Ｓ 被克隆到ｐ Ｍ Ｆ Ｔ７５ 载体上。将此质粒转化的大肠杆菌 Ｊ Ｍ１０９（ Ｄ Ｅ３） 中, 该转化子粗抽液的比活是宿主菌的２ ５００ 倍。通过 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ Ｆａｓｔ Ｆｌｏｗ 和 Ｂｌｕｅ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ６ Ｂ两步柱层析在一天内即可将精氨酰ｔ Ｒ Ｎ Ａ 合成酶纯化至电泳一条带, 比活为３６ ０００ ｕ／ｍｇ , 总收率可达６９ ％ 。与以前报道的 Ａｒｇ Ｒ Ｓ的高表达质粒相比, 使用该重组质粒可以很方便地将昂贵的标记氨基酸高效地参入酶分子内。目前的研究结果表明,该新系统能够很方便地提供大量的更高比活的大肠杆菌精氨酰ｔ Ｒ Ｎ Ａ 合成酶以进行该酶的 Ｎ Ｍ Ｒ 和结晶学研究     

Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.     

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.     

19F NMR of 5-fluorouracil-substituted transfer RNA transcribed in vitro: resonance assignment of fluorouracil-guanine base pairs.

5-Fluorouracil is readily incorporated into active tRNA(Val) transcribed in vitro from a recombinant phagemid containing a synthetic E. coli tRNA(Val) gene. This tRNA has the expected sequence and a secondary and tertiary structure resembling that of native 5-fluorouracil-substituted tRNA(Val), as judged by 19F NMR spectroscopy. To assign resonances in the 19F spectrum, mutant phagemids were constructed having base changes in the tRNA gene. Replacement of fluorouracil in the T-stem with cytosine, converting a FU-G to a C-G base pair, results in the loss of one downfield peak in the 19F NMR spectrum of the mutant tRNA(Val). The spectra of other mutant tRNAs having guanine for adenine substitutions that convert FU-A to FU-G base pairs all have one resonance shifted 4.5 to 5 ppm downfield. These results allow assignment of several 19F resonances and demonstrate that the chemical shift of the 19F signal from base-paired 5-fluorouracil differs considerably between Watson-Crick and wobble geometry.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.     

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.

Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.     

Analysis of the kinetic mechanism of arginyl-tRNA synthetase

A kinetic analysis of the arginyl-tRNA synthetase (ArgRS) from Escherichia coli was accomplished with the goal of improving the rate equations so that they correspond more closely to the experimental results. 22 different steady-state kinetic two-ligand experiments were statistically analysed simultaneously. A mechanism and values for the ArgRS constants were found where the average error was only 6.2% and ranged from 2.5 to 11.2% in the different experiments. The mechanism included not only the normal activation and transfer reactions but also an additional step which may be a conformational change after the transfer reaction but before the dissociation of the product Arg-tRNA from the enzyme. The forward rate constants in these four steps were low, 8.3-27 s(-1), but the reverse rate constants of the activation and transfer reactions were considerably higher (230 and 161 s(-1)). Therefore, in the presence of even low concentrations of PP(i) and AMP, the rate limitation occurs at the late steps of the total reaction. AMP increases the rate of the ATP-PP(i) exchange reaction due to the high reverse rate in the transfer reaction. The rate equation obtained was used to calculate the steady-state enzyme intermediate concentrations and rates between the intermediates. Three different Mg2+ binding sites were required to describe the Mg2+ dependence. One of them was the normal binding to ATP and the others to tRNA or enzyme. The measured Mg2+ dependence of the apparent equilibrium constant of the ArgRS reaction was consistent with the Mg2+ dependences of the reaction rates on the rate equation. Chloride inhibits the ArgRS reaction, 160 mM KCl caused a 50% inhibition if the ionic strength was kept constant with K-acetate. KCl strongly affected the K(m)(app) (tRNA) value. A difference was detected in the progress curves between the aminoacylation and ATP-PP(i) exchange rates. When all free tRNA(Arg) had been used from the reaction mixture, the aminoacylation reaction stopped, but the ATP-PP(i) exchange continued at a lowered rate.     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding.

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.     

The crystal structure of arginyl-tRNA synthetase from Homo sapiens

Arginyl-tRNA synthetase (ArgRS) is a tRNA-binding protein that catalyzes the esterification of l-arginine to its cognate tRNA. l-Canavanine, a structural analog of l-arginine, has recently been studied as an anticancer agent. Here, we determined the crystal structures of the apo, l-arginine-complexed, and l-canavanine-complexed forms of the cytoplasmic free isoform of human ArgRS (hArgRS). Similar interactions were formed upon binding to l-canavanine or l-arginine, but the interaction between Tyr312 and the oxygen of the oxyguanidino group was a little bit different. Detailed conformational changes that occur upon substrate binding were explained. The hArgRS structure was also compared with previously reported homologue structures. The results presented here may provide a basis for the design of new anticancer drugs, such as l-canavanine analogs.     

大肠杆菌精氨酰tRNA合成酶基因的诱导高表达

在高表达大肠杆菌精氨酰ｔＲＮＡ合成酶基因５５０倍的基础上,将ａｒｇ２的编码起始位点经基因突点突变导入ＮｃｏＩ限制性内切酶的位点后,重组到受异丙基硫代－β－Ｄ半乳糖苷诱导的ｐＴｒ９９Ｂ质粒上,使ａｒｇＳ比受体菌表达高近２０００倍。通过一步ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析则可得到ＳＤＳ－ＰＡＧＥ一条带的ＡｒｇＲＳ,比活为１５０００ｕ／ｍｇ,与文献相同。     

19F NMR detection of the complex between amantadine and the receptor portion of the influenza A M2 ion channel in DPC micelles

(19)F NMR probes were used to follow interactions between ligands in the aminoadamantane series, amantadine (Am) 1 and 3-F-Am 2, and the 5-F-Trp20 transmembrane fragment of the influenza A M2 proton channel (F-M2TM 3) in dodecylphosphocholine micelles over the pH range 5-8. Above pH 7, when the peptide adopts a tetrameric state that is able to bind channel blocking ligands, (19)F-Trp signals from both the free and bound states of the M2TM tetramer are resolved. This differentiation of bound and unbound states of the M2TM receptor by (19)F NMR may provide a system for SAR studies.     

Crystal structure of E. coli arginyl-tRNA synthetase and ligand binding studies revealed key residues in arginine recognition

The arginyl-tRNA synthetase (ArgRS) catalyzes the esterification reaction between L-arginine and its cognate tRNA^Arg. Previously reported structures of ArgRS shed considerable light on the tRNA recognition mechanism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS (eArgRS) complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry (ITC). Combined with previously reported work on ArgRS, our results elucidated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.     

The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties

Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.