title: Two homologous EF-G proteins from Pseudomonas aeruginosa exhibit distinct functions.
abstract: Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k cat) for the hydrolysis of GTP than EF-G1A; 0.2 s(-1) vs. 0.04 s(-1). These values resulted in specificity constants (k cat (obs)/K M) for EF-G1A and EF-G1B of 0.5 x 10(3) s(-1) M(-1) and 3.0 x 10(3) s(-1) M(-1), respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.
authors.affiliation: The University of Texas-Pan American, Edinburg, Texas, United States of America.
authors.forename: Stephanie O
authors.initials: SO
authors.lastname: Palmer
authors.forename: Edna Y
authors.initials: EY
authors.lastname: Rangel
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Hu
authors.forename: Alexis T
authors.initials: AT
authors.lastname: Tran
authors.forename: James M
authors.initials: JM
authors.lastname: Bullard
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D005672:Fusidic Acid / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D020653:Peptide Elongation Factor G / Q000235:genetics* / Q000378:metabolism*; D011072:Poly U / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D001426:Bacterial Proteins; D020653:Peptide Elongation Factor G; D011072:Poly U; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases