Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain.
The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
Jensen
M
M
Laboratoire de Biochimie, Centre National de la Recherche Scientifique no. 144, Ecole Polytechnique, Palaiseau, France.
Cool
R H
RH
Mortensen
K K
KK
Clark
B F
BF
Parmeggiani
A
A
D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000032:analysis; D006150:Guanine Nucleotides / Q000032:analysis*; D006153:Guanosine Diphosphate / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000032:analysis*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D010443:Peptide Chain Termination, Translational; D010444:Peptide Elongation Factor Tu / Q000032:analysis*; D011489:Protein Denaturation; D012343:RNA, Transfer / Q000032:analysis; D012270:Ribosomes / Q000201:enzymology; D013329:Structure-Activity Relationship
D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu