title: Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.
abstract: We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 μM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) μM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 μM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) μM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.
authors.affiliation: Chemistry Department, SCIE. 3.320, The University of Texas-Pan American, 1201 W. University Drive, Edinburg, TX 78541, USA.
authors.forename: Stephanie O
authors.initials: SO
authors.lastname: Palmer
authors.forename: Edna Y
authors.initials: EY
authors.lastname: Rangel
authors.forename: Alberto E
authors.initials: AE
authors.lastname: Montalvo
authors.forename: Alexis T
authors.initials: AT
authors.lastname: Tran
authors.forename: Kate C
authors.initials: KC
authors.lastname: Ferguson
authors.forename: James M
authors.initials: JM
authors.lastname: Bullard
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D010445:Peptide Elongation Factors; C023512:elongation factor Ts; D006153:Guanosine Diphosphate; D011072:Poly U; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu