Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis

Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp). It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp). Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp). These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp). The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp).     

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-specific differences in the regulation of the aminoacylation activity of mammalian tryptophanyl-tRNA synthetases

Tryptophanyl-tRNA synthetases (TrpRSs) catalyze the aminoacylation of tRNA^Trp. Previously, I demonstrated that Zn²⁺-depleted human TrpRS is enzymatically inactive and that binding of Zn²⁺ or heme to human TrpRS stimulates its aminoacylation activity. In the present study, bovine and mouse TrpRSs were found to be constitutively active regardless of the presence of Zn²⁺ or ferriprotoporphyrin IX chloride. Mutagenesis experiments demonstrated that the human H130R mutant is constitutively active and that the bovine R135H, E438A double mutant binds with Zn²⁺ or heme to enhance its aminoacylation activity as does human wild-type TrpRS. These results provide the first evidence of species-specific regulation of TrpRS activity.     

Indirect readout of tRNA for aminoacylation

Aminoacylation of tRNA by aminoacyl-tRNA synthetases is the essential reaction that matches protein amino acids with the trinucleotide sequences specified in mRNA. Direct electrostatic interactions made by tRNA synthetases with discriminating functional groups on the tRNA bases have long been known to determine aminoacylation specificity. However, structural and biochemical studies have revealed a second "indirect readout" mechanism that makes an important contribution as well. In indirect readout, the sequence-dependent conformations of tRNA are recognized through protein contacts with the sugar-phosphate backbone and with nonspecific portions of the bases. This mechanism appears to function in single-stranded regions, in canonical A-type duplex segments, and in the complex tertiary core portion of the tRNA. Operation of the indirect mechanism is not exclusive of the direct mechanism, and both are further mediated by induced-fit rearrangements, in which enzyme and tRNA undergo precise conformational changes after formation of an initial encounter complex. The examples of indirect readout in tRNA synthetase complexes extend the concept beyond its traditional application to DNA duplexes and serve as models for the operation of this mechanism in more complex systems such as the ribosome.     

Origin of the genetic code and specificity of tRNA aminoacylation. A testable model

Conclusion The specificity of tRNA aminoacylation as well as the origin of the genetic code are far from being understood at the molecular and evolutionnary level. The tRNA-tRNA interaction model could provide a missing link for resolving both problems. The model suggests a direct chemical interaction between the nucleotides in the anticodon, and the amino acid (adenylate) to be transferred to the 3-terminal adenosine, within the catalytic center (23). The experimental data reviewed here indicate that in many, but not all, systems the anticodon does play a major role during the aminoacylation and that the simultaneous binding of two tRNA molecules for aminoacylation (of only one of them) does not contradict enzymatic and crystallographic data (24).     

Identification of essential domains for Escherichia coli tRNA(leu) aminoacylation and amino acid editing using minimalist RNA molecules

Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNA(leu) analog that has been aminoacylated to a significant extent to date. A series of tRNA(leu) analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem-loop and variable arm stem-loop, but neither the D-arm nor T-arm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA. The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase. The anticodon and variable stem-loop domains were also dispensable for hydrolysis of the charged tRNA(leu) mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.     

Conservation of a tRNA core for aminoacylation

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.     

Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems

The tRNA identity rules ensuring fidelity of translation are globally conserved throughout evolution except for tyrosyl-tRNA synthetases (TyrRSs) that display species-specific tRNA recognition. This discrimination originates from the presence of a conserved identity pair, G1-C72, located at the top of the acceptor stem of tRNA(Tyr) from eubacteria that is invariably replaced by an unusual C1-G72 pair in archaeal and eubacterial tRNA(Tyr). In addition to the key role of pair 1-72 in tyrosylation, discriminator base A73, the anticodon triplet and the large variable region (present in eubacterial tRNA(Tyr) but not found in eukaryal tRNA(Tyr)) contribute to tyrosylation with variable strengths. Crystallographic structures of two tRNA(Tyr)/TyrRS complexes revealed different interaction modes in accordance with the phylum-specificity. Recent functional studies on the human mitochondrial tRNA(Tyr)/TyrRS system indicates strong deviations from the canonical tyrosylation rules. These differences are discussed in the light of the present knowledge on TyrRSs.     

Alternative design of a tRNA core for aminoacylation

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.     

Effects of spermine and magnesium ions on the aminoacylation of yeast tRNA(Tyr)

Stimulatory effects of Mg2+ and spermine on the kinetics of the aminoacylation of tRNA(Tyr) were examined using purified yeast tRNA(Tyr) and tyrosyl-tRNA synthetase. The apparent Km for tRNA(Tyr) was the lowest at Mg2+ concentrations between 2 and 5 mM and was not influenced by spermine. In the absence of spermine, the apparent Vmax was the highest at Mg2+ concentrations of 5 mM or higher, whereas the presence of spermine strongly stimulated the reaction at lower Mg2+ concentrations. Spermine alone could not substitute for Mg2+, nor was it able, at any Mg2+ concentration, to increase the reaction rate above the level reached at high concentrations of Mg2+ alone. Calculations of the concentration of Mg3.tRNA(Tyr) complex as a function of initial Mg2+ concentration, using the binding constants derived from physical measurements, allow the conclusion that spermine exerts its stimulatory activity by creating strong binding sites for Mg2+; this would enable the tRNA to assume the conformation required for optimal aminoacylation. The conformational requirement for the first tRNA: synthetase encounter is obviously less stringent, since the apparent Km for tRNA(Tyr) is not influenced by spermine.