Visualization of a Ternary Complex of the Escherichia coli Phe-tRNA Phe and Tu · GTP from Thermus thermophilus by Scanning Transmission Electron Microscopy

Abstract

Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T. thermophilus elongation factor (EF) Tu · GTP and the Escherichia coli Phe-tRNA Phe labeled with an undecagold (Au 11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47. The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles. Under conditions used for STEM imaging, the ternary complex is formed between Au 11-labeled Phe-tRNA Phe and Tu · GTP in a yield up to 25%. The stoichiometry of EF-Tu · GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu · GTP · aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism. The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu · GDP and nonaminoacylated tRNA Phe with Tu · GTP.