Cloning of a Dna Fragment Encoding γ-Glutamyl Kinase and γ-Glutamyl Phosphate Reductase from a Tomato cDNA Library
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We cloned the gene encoding the first enzyme of proline synthesis from tomato (L. esculentum) by complementation of a proB mutation in E. coli with a λgt-11 cDNA library of tomato fruit. We obtained seven phages which were able to restore proline prototrophy to the proB mutant. The insert from one of the complementing phages, PRO1, was subcloned into plasmid pBluescript IIKS+ When transferred into a mutant ΔproBA (which is deficient in both the first and second enzymes of proline biosynthesis, γ-glutamyl kinase and γ-glutamyl phosphate reductase), the PRO1 gene was able to complement this deletion mutation. Assays of the coupled γ-glutamyl kinase/γ-glutamyl phosphate reductase activity of E. coli proB or ΔproBA mutant strains revealed that these strains have elevated levels of both γ-glutamyl kinase and γ-glutamyl phosphate reductase. These results indicate that the PRO1 gene is a hybrid locus which specifies both enzymatic activities.
Nucleotide sequence analysis revealed that the 5’ portion of the PRO1 locus contains an 849 bp open reading frame which has the coding capacity for a protein with extensive amino acid sequence similarities to γ-glutamyl kinase from E. coli, yeast and Δ1-pyrroline-5-caroboxylate synthetase from moth bean, and the 3’ region specifies a 1287 bp open reading frame with the coding capacity for a protein with extensive similarity to γ-glutamyl phosphate reductase from E. coli and Δ1-pyrroline-S-carboxylate synthetase from moth bean. Surprisingly, there is a TAA translation termination codon within the PRO1 region between the open reading frames that encode γ-glutamyl kinase and γ-glutamyl phosphate reductase. The structure of the PRO1 mRNA, which resembles polycistronic messages in prokaryotes, is unusual in eukaryotes because of difficulties in continuing translation across translation termination codons. As expected, in E. coli, the TAA codon is recognized as a translation terminator, so that in this host, the PRO1 mRNA is translated into two proteins with apparent molecular masses of 30 and 45 kd. We do not know the nature of the translation product of the PRO1 gene in plants: whether only the 30 kd γ-glutamyl kinase encoded at the 5’ end of the mRNA is made, whether the downstream γ-glutamyl phosphate reductase is translated as a separate polypeptide as a result of internal reinitiation, or whether the translation product is a long hybrid protein composed of γ-glutamyl kinase/γ-glutamyl phosphate reductase, which arose as a result of editing the TAA codon from the mRNA, by splicing or skipping over it by translational frameshifting.
KeywordsProline Biosynthesis Proline Synthesis Ribosomal Frameshifting Moth Bean Translation Termination Codon
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