Michael Ibba

Aminoacyl-tRNA synthesis: divergent routes to a common goal

Aminoacyl-tRNAs are key components in protein synthesis. They are formed directly by correct acylation of tRNA (by aminoacyl-tRNA synthetases) or indirectly by tRNA-dependent transformation of misacylated tRNAs. The accuracy of aminoacyl-tRNA synthesis is enhanced by a number of further protein-RNA or protein-protein interactions, some of which are restricted to Archaea, and might reflect adaptation mechanisms to diverse conditions.

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl‐tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzymes active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS‐D235H resulted in a 4‐fold increase in the Km for the Gln, which was reduced to a 2‐fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3′ terminus. Disruption of these interactions can change the Km for Gln over a 30‐fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln‐GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein‐RNA interactions facilitate catalysis.

Transfer RNA recognition by class I lysyl-tRNA synthetase from the Lyme disease pathogen Borrelia burgdorferi

Borrelia burgdorferi and other spirochetes contain a class I lysyl‐tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNALys recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNALys in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNALys were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNAAsp scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.

Retracing the evolution of amino acid specificity in glutaminyl-tRNA synthetase

Molecular phylogenetic studies of glutaminyl‐tRNA synthetase suggest that it has relatively recently evolved from the closely related enzyme glutamyl‐tRNA synthetase. We have now attempted to retrace one of the key steps in this process by selecting glutaminyl‐tRNA synthetase mutants displaying enhanced glutamic acid recognition. Mutagenesis of two residues proximal to the active site, Phe‐90 and Tyr‐240, was found to improve glutamic acid recognition 3–5‐fold in vitro and resulted in the misacylation of tRNAGln with glutamic acid. In vivo expression of the genes encoding these misacylating variants of glutaminyl‐tRNA synthetase reduced cellular growth rates by 40%, probably as a result of an increase in translational error rates. These results provide the first biochemical evidence that glutaminyl‐tRNA synthetase originated through duplication and consequent diversification of an ancestral glutamyl‐tRNA synthetase‐encoding gene.

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNAGln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3′ end of tRNAGln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNATyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNACUAGln from tRNA2Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher Km for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.

Glutaminyl‐tRNA synthetase: from genetics to molecular recognition

Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code. They are synthesized by the aminoacyl‐tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations. Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl‐tRNA synthetase (GlnRS). Sequence‐specific interactions between GlnRS and tRNAGln determine both the accuracy of tRNA selection and the efficiency of aminoacylation. Thus, amino acid recognition is tRNA‐dependent. Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA–enzyme complex displays a considerably reduced affinity for glutamine compared to wild‐type. This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.

Recognition of One tRNA by Two Classes of Aminoacyl-tRNA Synthetase

Lysyl-tRNA synthetases are unique amongst the aminoacyl-tRNA synthetases in being composed of two unrelated families. In most bacteria and all eukarya, the known lysyl-tRNA synthetases are subclass He-type aminoacyl-tRNA synthetases whereas some archaea and bacteria have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. We have now examined substrate recognition by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase. The genes encoding both enzymes were able to rescue an Escherichia coli strain deficient in lysyl-tRNA synthetase, indicating their ability to functionally substitute for class II lysyl-tRNA synthetases in vivo. In vitro characterization revealed lysine activation and recognition to be tRNA-dependent, a phenomenon previously reported for other class I aminoacyl-tRNA synthetases. More detailed examination of tRNA recognition has shown that class I lysyl-tRNA synthetases recognize the same elements in tRNALys as their class II counterparts; specifically, the discriminator base (N73) and the anticodon serve as recognition elements. The implications of these results for the evolution of Lys-tRNALys synthesis and their possible indications of a more ancient origin for tRNA then aminoacyl-tRNA synthetases will be discussed.