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Arch Biochem Biophys. 1994 Jan;308(1):109-17.

Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.

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  • 1Ohio State Biochemistry Program, Ohio State University, Columbus 43210-1292.


Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea (Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

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