IDENTIFICATION OF THE ELONGATION-FACTOR TU BINDING-SITE ON 70S ESCHERICHIA-COLI RIBOSOMES BY CHEMICAL CROSS-LINKING

Elongation factor Tu (EF-Tu), in the presence of Phe-tRNA, GMPPCP, and Poly (U), binds to 70S ribosomes at the recognition (R) site. In orde r to identify the ribosomal proteins adjacent to the EF-Tu occupying t he R site, EF-Tu:Phe-tRNA:GMPPCP:ribosome complexes were crosslinked b y modification with 2-iminothiolane and mild oxidation to form disulfi de bridges between neighbouring proteins whose endogenous or introduce d SH groups were appropriately located. The binding of Phe-tRNA to the ribosome was shown to be largely dependent on the presence of Poly (U ). The total protein from the complexes was extracted and separated by two-dimensional gel electrophoresis by non-equilibrium pH gradient el ectrophoresis (NEpHGE) in the first dimension, followed by gradient SD S gel electrophoresis in the second dimension. Comparison of control s amples crosslinked without Poly (U) to those crosslinked with Poly (U) present showed a single crosslinked complex in the region of the gel near EF-Tu. No cross-links in the vicinity of EF-Tu were visible in th e absence of Poly (U). The crosslinked proteins in this region were re covered by electroelution, radiolabeled and their identity was confirm ed by 2D gel electrophoresis and immunoblot analyses. Two major 5.0S r ibosomal proteins, L7/L12 and L10 were found to be covalently linked t o EF-Tu. The isolated crosslinked complex did not contain any protein from the 30S subunit. These results demonstrate that L7/L12 and L10 ar e the major, if not only, ribosomal protein cross-links to EF-Tu in th e R site. In contrast to previous crosslinking results obtained by oth ers, our results define a unique location for the EF-Tu binding site, one compatible with functional data and near that of the EF-G binding site on the ribosome.