A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution

Abstract

Efforts to delineate the advent of many enzymes essential to protein translation are often limited by the fact that the modern genetic code evolved before divergence of the tree of life. Glutaminyl-tRNA synthetase (GlnRS) is one noteworthy exception to the universality of the translation apparatus. In eukaryotes and some bacteria, this enzyme is essential for the biosynthesis of Gln-tRNAGln, an obligate intermediate in translation. GlnRS is absent, however, in archaea, and most bacteria, organelles, and chloroplasts. Phylogenetic analyses predict that GlnRS arose from glutamyl-tRNA synthetase (GluRS), via gene duplication with subsequent evolution of specificity. A pertinent question to ask is whether, in the advent of GlnRS, a transient GluRS-like intermediate could have been retained in an extant organism. Here, we report the discovery of an essential GluRS-like enzyme (GluRS2), which coexists with another GluRS (GluRS1) in Helicobacter pylori. We show that GluRS2's primary role is to generate Glu-tRNAGln, not Glu-tRNAGlu. Thus, GluRS2 appears to be a transient GluRS-like ancestor of GlnRS and can be defined as a GluGlnRS.

Fig. 1.

A consensus model for the evolution of GlnRS and GluRS-D from an ancestral GluRS-ND (, –15, 19, 20). The dashed arrows represent evolutionary events relevant to this work (also, see Fig. 4B). The endpoints of these evolutionary paths are defined by the three modern enzymes (GluRS-ND, GluRS-D, and GlnRS), still found in extant organisms. Bs, B. subtilis; Ec, E. coli; Hs, Homo sapiens; gltX-D (GluRS-D), discriminating GluRS; gltX-ND (GluRS-ND), nondiscriminating GluRS.

Fig. 4.

Evolution of bacterial GluRSs. (A) Distance tree of bacterial GluRS, GluRS1, and GluRS2 sequences. An ancient gene duplication event (*) separates GluRS1 (dark gray) and GluRS2 (light gray) sequences in the proteobacteria. The genomes of organisms labeled with bullets (•) contain a glnS ORF. (B) Model for the evolution of GluRS in the proteobacteria. Dashed arrows indicate possible future events in evolution of tRNA specificities. See Materials and Methods for tree derivations.

Fig. 2.

Overexpression of GluRS2 induces a toxic growth phenotype in E. coli, caused by misacylation of E. coli tRNA^Gln1. (A) E. coli DH5α was transformed with pSS001 (GluRS1, □), pSS002 (GluRS2, •), or unmodified pQE-80 (○). Cultures were grown in LB, supplemented with 100 μg/ml ampicillin. Growth was monitored at 600 nm. After each growth, pSS001 and pSS002 were purified and each insert was resequenced to verify that random mutations did not occur. Similar results were obtained by using E. coli K-12 (data not shown). (B) Acid gel and Northern blot analysis of E. coli tRNA^Gln1 misacylation by H. pylori GluRS2. Hybridization was conducted with an E. coli tRNA^Gln1-specific oligonucleotide (Table 2); the oligonucleotide was kinased with [γ-³²P]ATP before hybridization.

Fig. 3.

Transfer RNA specificities of GluRS1 and GluRS2. (A) Glu-tRNA^AA biosynthesis by GluRS1 (black) versus GluRS2 (red). (Top) tRNA^Glu1.(Middle) tRNA^Glu2. (Bottom) tRNA^Gln. Assays shown are the average of triplicate experiments (see Materials and Methods). (B) Acid gel and Northern blot analysis of H. pylori tRNA^Glu1 (Top), tRNA^Glu2 (Middle), and tRNA^Gln (Bottom). The black line in the middle of the bottom two gels represents empty gel lanes that were removed for clarity. Each tRNA/GluRS combination was evaluated individually (lanes 2 and 3) and as a mixture of tRNAs (lanes 4 and 5). For comparison, lane 1 shows each tRNA preparation in the deacylated form, before incubation with either GluRS1 or GluRS2. (C) Glutamine is not a substrate for GluRS2. A standard aminoacylation assay was performed (see Materials and Methods) with GluRS2, 2 μM tRNA^Gln, and 100 μM glutamate (□) or 100 μM glutamine (▪). The graph represents averaged data from experiments run in triplicate.