Purification and N-Terminal Sequence-Analysis of Pea Chloroplast Protein Synthesis Factor EF-G

• Published in: Archives of biochemistry and biophysics Vol. 308; no. 1; pp. 109 - 117
• Main Authors: Akkaya, M.S., Welcsh, P.L., Wolfe, M.A., Duerr, B.K., Becktel, W.J., Breitenberger, C.A.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 1994; Elsevier
• Subjects: Amino Acid Sequence; Animals; Arabidopsis thaliana; Base Sequence; Biological and medical sciences; Blotting, Southern; CHLOROPLASTE; Chloroplasts - metabolism; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cloning, Molecular; CLOROPLASTO; DNA - isolation & purification; DNA - metabolism; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - metabolism; Fabaceae - metabolism; Fundamental and applied biological sciences. Psychology; Fusidic Acid - pharmacology; Genes, Plant; Light; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Peptide Elongation Factor G; Peptide Elongation Factors - biosynthesis; Peptide Elongation Factors - chemistry; Peptide Elongation Factors - isolation & purification; PISUM SATIVUM; Plants, Medicinal; PLANTULAS; PLANTULE; Polymerase Chain Reaction; PROTEINAS; PROTEINE; PURIFICACION; PURIFICATION; Rats; Restriction Mapping; Ribosomes - metabolism; SECUENCIA NUCLEICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEIQUE; SINTESIS DE PROTEINAS; SYNTHESE PROTEIQUE; Translation. Translation factors. Protein processing; Purification; Elongation factor EFG; Sequence alignment; Leguminosae; Protein synthesis; Dicotyledones; Pisum sativum; Angiospermae; N terminal-Sequence; Spermatophyta; Elongation; Chloroplast; Aminoacid sequence; Structural analysis
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1994.1016

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.