The state of aggregation of elongation factor Tu from E. coli : from monomers to polymers

Abstract

Elongation factor Tu is a 43,200 MW protein from prokaryotes which is known to carry aminoacyl-tRNAs to the mRNA-programmed ribosome. The state of aggregation of elongation factor Tu (EF-Tu), purified to near homogeneity from E. Coli, has been examined here under various conditions in vitro. At pH 7.5, EF-Tu appears to be mostly a monomer to time-resolved fluorescence spectroscopy experiments, but quasi-elastic light scattering data suggests the presence of a small amount of higher aggregates. Polymerization of EF-Tu has been shown to be induced by pH below 7 under low ionic strength conditions. Polymerization curves, as monitored by turbidity, show a lag time followed by rapid growth in a fashion similar to that for actin and tubulin. However, the inability to seed polymerization with preformed polymers suggests that polymerization of EF-Tu does not proceed via the canonical nucleation/elongation model. Polymerization was shown to be inhibited by pH above 7, high ionic strength, particularly magnesium and calcium ions, low temperature and the presence of aurodox and nocodazole. Increasing concentrations up to 2.5 µM increase the rate of polymerization, but further increases to 9.8 µM decrease the rate of polymerization and increase the lag time. Such behavior with concentration is unique compared to other polymerizing systems. Also, polymerization of EF-Tu does not require bound GTP, as is the case with tubulin. Time-resolved fluorescence polarization studies on EF-Tu covalently labeled with 2,5-dansyl chloride indicate that the monomer concentration decreases as polymerization proceeds. The structure of the polymer formed by EF-Tu was examined by various techniques. Scanning and transmitting electron micrographs of EF-Tu polymerized at low pH show evidence of linear filament bundles, branched polymers and two-dimensional sheets. Light microscopy using fluorescence optics of EF-Tu labeled with tetramethylrhodamine isothiocyanate provided evidence for branched polymers similar in size (10-15 urn in diameter) to those observed under the electron microscope. Polymers of EF-Tu approximately 120nm in diameter behave as spheres, whereas smaller polymers behave more as linear rods which show almost no interaction. An interesting observation was that monomers added to a solution of polymers tend to form new polymers instead of adding onto pre-formed polymers, which is exactly opposite to the behavior of known polymerizing proteins and suggests a size limit. Finally, time-resolved phosphorescence anisotropy of EF-Tu labeled with erythrosin isothiocyanate revealed that as polymers grow, they become entangled. There was a fast rotational motion which was resolved by increasing the solvent viscosity with glycerol. The sub-microsecond motion may be due to either internal flexibility of the polymers or the presence of small aggregates such as hexamers or heptamers.