Critical residues for RNA discrimination of the histone hairpin binding protein (HBP) investigated by the yeast three-hybrid system

The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3' UTR of the cell-cycle-dependent histone mRNAs. HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain. The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure. By means of negative selections followed by positive selections, we isolated mutant HBP species. Our results indicate tight relationships between the RBD and the N- and C-terminal domains.     

Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002.

The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS). The gene coding for this enzyme was isolated from E. coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector. Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence. As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E. coli JM103, about 100 times as much modified ArgRS. This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered. The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process.     

Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.     

Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.     

Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding

The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA(Arg) by yeast arginyl-tRNA synthetase. Alanine mutagenesis was used to probe all the side chain mediated interactions that occur between tRNA(Arg2)(ICG) and ArgRS. The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA(Arg) isoacceptors. Nine mutants that generate lethal phenotypes were identified, suggesting that only a limited set of side chain mediated interactions is essential for tRNA recognition. The majority of the lethal mutants was mapped to the anticodon binding domain of ArgRS, a helix bundle that is characteristic for class Ia synthetases. The alanine mutations induce drastic decreases in the tRNA charging rates, which is correlated with a loss in affinity in the catalytic site for ATP. One of those lethal mutations corresponds to an Arg residue that is strictly conserved in all class Ia synthetases. In the known crystallographic structures of complexes of tRNAs and class Ia synthetases, this invariant Arg residue stabilizes the idiosyncratic conformation of the anticodon loop. This paper also highlights the crucial role of the tRNA and enzyme plasticity upon binding. Divalent ions are also shown to contribute to the induced fit process as they may stabilize the local tRNA-enzyme interface. Furthermore, one lethal phenotype can be reverted in the presence of high Mg(2+) concentrations. In contrast with the bacterial system, in yeast arginyl-tRNA synthetase, no lethal mutation has been found in the ArgRS specific domain recognizing the Dhu-loop of the tRNA(Arg). Mutations in this domain have no effects on tRNA(Arg) aminoacylation, thus confirming that Saccharomyces cerevisiae and other fungi belong to a distinct class of ArgRS.     

Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases

Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.     

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.     

The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation

Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS.