Protein biosynthesis in organelles requires misaminoacylation of tRNA

In the course of our studies on transfer RNA involvement in chlorophyll biosynthesis1, we have determined the structure of chloroplast glutamate tRNA species. Barley chloroplasts contain in addition to a tRNAGlu species at least two other glutamate-accepting tRNAGlus. We now show that the sequences of these tRNAs differ significantly: they are differentially modified forms of tRNAGln (as judged by their UUG anticodon). These mischarged Glu-tRNAGln species can be converted in crude chloroplast extracts to Gln-tRNAGln. This reaction requires a specific amidotransferase and glutamine or asparagine as amide donors. Aminoacylation studies show that chloroplasts, plant and animal mitochondria, as well as cyanobacteria, lack any detectable glutaminyl-tRNA syn-thetase activity. Therefore, the requirement for glutamine in protein synthesis in these cells and organelles is provided by the conversion of glutamate attached to an 'incorrectly' charged tRNA. A similar situation has been described for several species of Gram-positive bacteria2. Thus, it appears that the occurrence of this pathway of Gln-tRNAGln formation is widespread among organisms and is a function conserved during evolution. These findings raise questions about the origin of organelles and about the evolution of the mechanisms maintaining accuracy in protein biosynthesis.

[1]  D Sankoff,et al.  On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. , 1984, Nucleic acids research.

[2]  G. Burkard,et al.  Comparative studies of the tRNA's and the aminoacyl-tRNA synthetases from the cytoplasm and the chloroplasts of Phaseolus vulgaris. , 1970, Biochimica et biophysica acta.

[3]  M. Wilcox.  γ-Glutamyl Phosphate Attached to Glutamine-Specific tRNA , 1969 .

[4]  T. Kohchi,et al.  Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA , 1986, Nature.

[5]  M. Wilcox,et al.  Transfer RNA as a cofactor coupling amino acid synthesis with that of protein. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Steinmetz,et al.  Hybridization of bean, spinach, maize and Euglena chloroplast transfer RNAs with homologous and heterologous chloroplast DNAs. An approach to the study of homology between chloroplast tRNAs from various species. , 1980, Biochimica et biophysica acta.

[7]  B. A. Roe,et al.  Studies on human tRNA. I. The rapid, large scale isolation and partial fractionation of placenta and liver tRNA , 1975, Nucleic Acids Res..

[8]  E. V. Arx,et al.  Eine Multidimensionale technik zur chromatographischen identifizierung von aminosäuren , 1963 .

[9]  R. Gupta.  Halobacterium volcanii tRNAs. Identification of 41 tRNAs covering all amino acids, and the sequences of 33 class I tRNAs. , 1984, The Journal of biological chemistry.

[10]  B. Ledwith,et al.  Characterization of a DNA primase from rat liver mitochondria. , 1986, The Journal of biological chemistry.

[11]  D. Caput,et al.  Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Peattie,et al.  Direct chemical method for sequencing RNA. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Stanley,et al.  A different approach to RNA sequencing , 1978, Nature.

[14]  S. Bernhard,et al.  Metabolite transfer via enzyme-enzyme complexes. , 1986, Science.

[15]  H. Fukuhara,et al.  Isoaccepting mitochondrial glutamyl-tRNA species transcribed from different regions of the mitochondrial genome of Saccharomyces cerevisiae. , 1976, Journal of molecular biology.

[16]  D. Hall,et al.  Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation. , 1979, Plant physiology.

[17]  C R Woese,et al.  Mitochondrial origins. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Söll,et al.  The RNA required in the first step of chlorophyll biosynthesis is a chloroplast glutamate tRNA , 1986, Nature.

[19]  F. Jurnak,et al.  Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP. , 1984, The Journal of biological chemistry.

[20]  Département de Biochimie,et al.  A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro , 1986, Journal of bacteriology.

[21]  S. Rasmussen,et al.  Biosynthesis of Δ-aminolevulinate in greening barley leaves VI. Activation of glutamate by ligation to RNA , 1984 .