A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.

Abstract

The universal precursor of tetrapyrrole pigments (e.g., chlorophylls and hemes) is 5-aminolevulinic acid (ALA), which in Euglena gracilis chloroplasts is derived via the two-step C5 pathway from glutamate charged to tRNAGIW. The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNAGu (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNAGIu. The second enzyme, GSA-2,1-aminomutase, converts GSA to ALA. tRNAGIu is a specific cofactor for the NADPH-dependent reduction by GluTR, an enzyme that recognizes the tRNA in a sequence-specific manner. This RNA is the normal tRNAGUI, a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (YgZNaIL)

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…Base-specific requirements imposed by glutamyl-tRNA reductase on tRNA"'" have recently been determined for Euglena gracilis (Stange-Thomann et al, 1994). In a yellow mutant of this alga, a single point mutation in tRNA"'" was found to uncouple chlorophyll and plastid protein synthesis (Stange-Thomann et al, 1994). The tRNAG'" could still be charged by glutamyl-tRNA synthetase but was no longer able to participate in the glutamyl-tRNA reductase reaction.…”

“…Barley glutamyl-tRNA reductase also did not use cytosolic glutamyl-tRNAG'" in a homologous in vitro system (Peterson et al, 1988), which demonstrates its high tRNA specificity. Base-specific requirements imposed by glutamyl-tRNA reductase on tRNA"'" have recently been determined for Euglena gracilis (Stange-Thomann et al, 1994). In a yellow mutant of this alga, a single point mutation in tRNA"'" was found to uncouple chlorophyll and plastid protein synthesis (Stange-Thomann et al, 1994).…”

“…The tRNAG'" could still be charged by glutamyl-tRNA synthetase but was no longer able to participate in the glutamyl-tRNA reductase reaction. There appear to be unique bases also in higher plant tRNA"'" that may serve as recognition (identity) elements for glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and/or the elongation factor EF-Tu Stange-Thomann et al, 1994).…”

Genetic control of chlorophyll metabolism

“…На первом этапе С GluRS) Активируя глутамат, тРНК Глу запускает биосинтез тетрапирролов (Huang et al, 1984). Её критическая роль в формировании ХЛ была показана при изучении бесхлорофильного оранжевого мутанта эвглены, у ко-торого причиной отсутствия пигмента оказалась точ-ковая мутация -замена (С-56-U) в Т-петле тРНК GLU (Stange-Thomann et al, 1994). Гены тРНК Глу у всех фо-тосинтезирующих эукариот консервативны и локали-зованы в хлоропластной ДНК (хлДНК).…”

tRNA Recognition by Glutamyl-tRNA Reductase

“…A point mutation (C56 -U56) in Euglena gracilis chloroplast tRNA Glu was reported to uncouple protein and chlorophyll biosynthesis. This mutant tRNA was still aminoacylated by chloroplast GluRS and utilized in protein biosynthesis but was not a substrate for GluTR and therefore did not support tetrapyrrole biosynthesis (15). The solution of the long sought-after crystal structure of GluTR (30) and the biochemical characterization of E. coli GluTR (16,17) generated much interest in the details of tRNA recognition by this protein.…”