Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.
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.
Akkaya
M S
MS
Ohio State Biochemistry Program, Ohio State University, Columbus 43210-1292.
Welcsh
P L
PL
Wolfe
M A
MA
Duerr
B K
BK
Becktel
W J
WJ
Breitenberger
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002736:Chloroplasts / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007887:Fabaceae / Q000378:metabolism*; D005672:Fusidic Acid / Q000494:pharmacology; D017343:Genes, Plant; D008027:Light; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D051381:Rats; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D005672:Fusidic Acid; D004247:DNA