NUCLEOTIDES OF TRANSFER-RNA (GLU) INVOLVED IN RECOGNITION BY BARLEY CHLOROPLAST GLUTAMYL-TRANSFER-RNA SYNTHETASE AND GLUTAMYL-TRANSFER-RNA REDUCTASE

The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex w ith the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the rec ognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a n umber of species were used to determine the nucleotides involved in th e recognition of the barley chloroplast tRNA(Glu) by the barley enzyme s. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The che mically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA s ynthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chl oroplasts of barley, Chlamydomonas reinhardtii, tobacco, cucumber, whe at and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia c oli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 con served bases and five of these bases, G(10), A(26), U-34, U-35 and A(3 7) are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(G lu) from E. coli, bakers yeast and barley germ and the barley chloropl ast glutamyl-tRNA(Gln) are not effective substrates for the barley chl oroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences o f these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yiel ds seven common differences in the primary sequence. These 7 nucleotid es, A(7)-U-66, U-29-A(41), A(53)-U-61, and U-72 are expected to be req uired for recognition by the barley chloroplast glutamyl-tRNA(Glu) red uctase.