Abstract
The expression of the Rhizobium meliloti glutamyl-tRNA synthetase gene in Escherichia coli under the control of a trc promoter results in a toxic effect upon isopropyl-beta-D-thiogalactopyranoside induction, which is probably caused by a misacylation activity. To further investigate this unexpected result, we looked at the pathway of Gln-tRNAGln formation in R. meliloti. No glutaminyl-tRNA synthetase activity has been found in R. meliloti crude extract, but we detected a specific aminotransferase activity that changes Glu-tRNAGln to Gln-tRNAGln. Our results show that R. meliloti, a member of the alpha-subdivision of the purple bacteria, is the first Gram-negative bacteria reported to use a transamidation pathway for Gln-tRNAGln synthesis. A phylogenetic analysis of the contemporary glutamyl-tRNA synthetase and glutaminyl-tRNA synthetase amino acid sequences reveals that a close evolutionary relationship exists between R. meliloti and yeast mitochondrial glutamyl-tRNA synthetases, which is consistent with an origin of mitochondria in the alpha-subdivision of Gram-negative purple bacteria. A 256-amino acid open reading frame closely related to bacterial glutamyl-tRNA synthetases, which probably originates from a glutamyl-tRNA synthetase gene duplication, was found in the 4-min region of the E. coli chromosome. We suggest that this open reading frame is a relic of an ancient transamidation pathway that occurred in an E. coli ancestor before the horizontal transfer of a eukaryotic glutaminyl-tRNA synthetase (Lamour, V., Quevillon, S., Diriong, S., N'Guyen, V. C., Lipinski, M., and Mirande, M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8670-8674) and that it favored its stable acquisition. From these observations, a revisited model for the evolution of the contemporary glutamyl-tRNA synthetases and glutaminyl-tRNA synthetases that differs from the generally accepted model for the evolution of aminoacyl-tRNA synthetases is proposed.
Highlights
The expression of the Rhizobium meliloti glutamyltRNA synthetase gene in Escherichia coli under the control of a trc promoter results in a toxic effect upon isopropyl--D-thiogalactopyranoside induction, which is probably caused by a misacylation activity
No R. meliloti GlnRS activity was eluted from a MonoQ column (Fig. 1) or was detected in an S-20 cell extract (Fig. 2) using either unfractionated E. coli or R. meliloti tRNA, whereas GluRS activity was detected in both cases
Since this misacylating activity of R. meliloti GluRS toward E. coli tRNA probably leads to errors in protein biosynthesis, it explains the in vivo toxic effect observed following expression of the R. meliloti gltX gene in E. coli
Summary
Based on tRNA discrimination properties of Escherichia coli GluRS and GlnRS (Rogers and Soll, 1993) and on the difference in tRNAGln aminoacylation pathways between Gram-negative and Gram-positive bacteria, an evolutionary scenario, consistent with the horizontal gene transfer hypothesis (Lamour et al, 1994), has been proposed by Rogers and Soll (1995). In their model, they suggest that the divergence between eukaryotic and bacterial “GlxRS” (an ancestral enzyme able to recognize all tRNA isoacceptors) reflects the process of accommodation to multiple tRNA isoacceptors (with anticodons (c/u)UX for eukaryotes and UUX for bacteria). It is in contrast to the generally accepted hypothesis suggesting that aaRS evolved to include the full set of 20-amino acid specificity before the divergence leading to prokaryotes and eukaryotes
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