Abstract
During the first step of porphyrin biosynthesis in Archaea, most bacteria, and in chloroplasts glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Elements in tRNA(Glu) important for utilization by Escherichia coli GluTR were determined by kinetic analysis of 51 variant transcripts of E. coli Glu-tRNA(Glu). Base U8, the U13*G22**A46 base triple, the tertiary Watson-Crick base pair 19*56, and the lack of residue 47 are required for GluTR recognition. All of these bases contribute to the formation of the unique tertiary core of E. coli tRNA-(Glu). Two tRNA(Glu) molecules lacking the entire anticodon stem/loop but retaining the tertiary core structure remained substrates for GluTR, while further decreasing tRNA size toward a minihelix abolished GluTR activity. RNA footprinting experiments revealed the physical interaction of GluTR with the tertiary core of Glu-tRNA(Glu). E. coli GluTR showed clear selectivity against mischarged Glu-tRNA(Gln). We concluded that the unique tertiary core structure of E. coli tRNA(Glu) was sufficient for E. coli GluTR to distinguish specifically its glutamyl-tRNA substrate.
Highlights
During the first step of porphyrin biosynthesis in Archaea, most bacteria, and in chloroplasts glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde
The earlier studies on Glutamyl-tRNA synthetase (GluRS) identity presented a basis for comparison and provided the large set of in vitro synthesized Glu-tRNAGlu variants required to identify residues in E. coli tRNAGlu that are important for E. coli GluTR activity
Specific recognition of Glu-tRNA by E. coli GluTR is brought about by the unique tertiary core of tRNAGlu created by the U13*G22**A46 base triple, ⌬47, and the tertiary base pair G19*C56
Summary
GluRS, glutamyl-tRNA synthetase; CD, circular dichroism; GSA, glutamat-1-semialdehyde; GSA-AM, glutamate-1-semialdehyde-2,1-aminomutase; GluTR, glutamyl-tRNA reductase; HPLC, high performance liquid chromatography; T7 RNAP, T7 RNA polymerase; N*N, secondary base pair; N**N, tertiary base pair. To maintain the fidelity of protein biosynthesis and, at the same time, an adequate metabolite flux for 5-aminolevulinic acid formation, accurate recognition of various cellular tRNAs by their cognate aminoacyl-tRNA synthetase and of Glu-tRNA by GluTR must be secured. Based on a sequence comparison of tRNAGlu species that are substrates for various GluTRs with tRNA species which are not utilized, putative identity elements were postulated [14]. A point mutation (C56 –U56) in Euglena gracilis chloroplast tRNAGlu 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 did not support tetrapyrrole biosynthesis [15]. This paper is available on line at http://www.jbc.org tRNA Recognition by Glutamyl-tRNA Reductase
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