Search

The canonical function of glutamyl‐tRNA synthetase (GluRS) is to glutamylate tRNAGlu. Yet not all bacterial GluRSs glutamylate tRNAGlu; many glutamylate both tRNAGlu and tRNAGln, while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu. Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx‐specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx‐specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu‐bound GluRS were from non‐proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non‐proteobacterial GluRS structures. Marked differences between proteobacterial and non‐proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx‐specificity vis‐a‐vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo‐ and non‐proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx‐specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D‐helix contacting loop of GluRS in the modulation of tRNAGln‐specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx‐specificity of GluRS, this is the first report of complementary signatures of tRNAGlx‐specificity in GluRS. [ABSTRACT FROM AUTHOR]

The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylum-specific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu-GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported. [ABSTRACT FROM AUTHOR]

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNA Glu . Yet not all bacterial GluRSs glutamylate tRNA Glu ; many glutamylate both tRNA Glu and tRNA Gln , while some glutamylate only tRNA Gln and not the cognate substrate tRNA Glu . Understanding the basis of the unique specificity of tRNA Glx is important. Mutational studies have hinted at hotspot residues, both on tRNA Glx and GluRS, which play crucial roles in tRNA Glx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNA Glx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNA Glu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNA Glx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNA Glx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNA Glx D-helix contacting loop of GluRS in the modulation of tRNA Gln -specificity. While earlier studies have identified functional hotspots on tRNA Glx that control the tRNA Glx -specificity of GluRS, this is the first report of complementary signatures of tRNA Glx -specificity in GluRS., (© 2023 Wiley Periodicals LLC.)