A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.
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 tRNA(Glu). The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA(Glu) (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNA(Glu). The second enzyme, GSA-2,1-aminomutase, converts GSA to ALA. tRNA(Glu) 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 tRNA(Glu), a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (Y9ZNalL) does not synthesize ALA from glutamate, although it contains GluTR and GSA-2,1-aminomutase activity. The tRNA(Glu) isolated from the mutant can still be acylated with glutamate in vitro and in vivo. Furthermore, it supports chloroplast protein synthesis; however, it is a poor substrate for GluTR. Sequence analysis of the tRNA and of its gene revealed a C56-->U mutation in the resulting gene product. C56 is therefore an important identity element for GluTR. Thus, a point mutation in the T loop of tRNA uncouples protein from chlorophyll biosynthesis.
Stange-Thomann
N
N
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114.
Thomann
H U
HU
Lloyd
A J
AJ
Lyman
H
H
Söll
D
D
D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002734:Chlorophyll / Q000096:biosynthesis*; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D005056:Euglena gracilis / Q000235:genetics / Q000378:metabolism*; D019751:Intramolecular Transferases; D007535:Isomerases / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012352:RNA, Transfer, Glu / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D012352:RNA, Transfer, Glu; D002734:Chlorophyll; D004247:DNA; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase