Cross-linking of Selected Residues in the N- and C-terminal Domains of Escherichia coli Protein L7/L12 to Other Ribosomal Proteins and the Effect of Elongation Factor Tu

Abstract

The Escherichia coli ribosomal protein, L7/L12, is the most extensively investigated representative of the small, four-copy, dimeric acidic proteins that are found in large ribosomal subunits of all organisms and exist as a conserved quaternary structural element in which two dimers are integrated into the ribosome through binding to a common anchoring protein (1-3). One or both of the L7/L12 dimers form a conspicuous morphological feature on the ribosome known in E. coli as the L7/L12 stalk (4). The proteins can be simply and selectively removed from and reconstituted into the ribosome (5), a property that greatly facilitates experiments of the type reported here in which genetically and biochemically modified proteins replace the wild type.The L7/L12 polypeptide is composed of two distinct organized structural domains linked by a flexible hinge (6, 7), as summarized in Fig. 1. The elongated, helical N-terminal domain, residues 1-33, is responsible for dimer interaction (8), and the globular C-terminal domain, residues 53-120, is necessary for factor binding (9 -11). The C-terminal domains can be cross-linked to each other in different orientations yet retain full functional activity in supporting polypeptide synthesis (12). Flexibility of L7/L12 has been demonstrated in solution by fluorescence techniques (13) and in the ribosome by proton NMR (14, 15), by electron microscopy (16), and with fluorescence probes attached to the C-terminal domains (13,17,18).Immunoelectron microscopy with monoclonal antibodies (19) directly showed the presence of the C-terminal domain at the tip of the stalk and the N-terminal domain at the base of the stalk. This was consistent with earlier demonstrations that the N-terminal domain was responsible for binding of the fulllength L7/L12 to L10 and to the ribosome (9, 10). It was shown that one dimer per particle was sufficient to form a visible stalk (20), despite earlier studies with polyclonal antibodies that had suggested that both dimers were present in the stalk (21). Different studies indicated that the presence of one L7/L12 dimer on the body of the 50 S particle in an extended conformation directed toward the central protuberance (22, 23). Additional evidence that a C-terminal domain can occupy a location not only extended across the body of the ribosome but also near the base of the stalk came from cross-linking between a predetermined location in the C-terminal domain, 25) and also from hinge deletion studies (26,27). A different heterobifunctional reagent, APDP 1 showed a cross-link between Cys-89 and L11 and also with L10 in a lesser extent near the EF-G binding site near the base of the stalk (28). The site-specific cross-linking experiments led to the proposal of a possible "bent" conformation for one of the dimers in which the C-terminal domain could lie on the body of the subunit near the N-terminal domain at the base of the stalk. We asked the question whether there was a preferred surface of the C-terminal domain that made these contacts and designed cysteine prob...

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

“…This observation is consistent with a previous finding of a covalent cross-link between L7/L12 and the protein L5 (38). This cross-link has been established both in the presence and absence of EF-G. For L7/L12 to bind at this location at least one dimer of the L7/L12 tetramer would have to be in an extended conformation, lying across the body of the 50 S subunit (39). By contrast the presence of individual proteins in the fusidic acid-inhibited complex indicates that a significant population of the stalk proteins is less constrained and therefore free to dissociate into individual monomers.…”

Interactions of an EssentialBacillus subtilisGTPase, YsxC, with Ribosomes

“…On the basis of immune electron microscopy, the two L7/L12 dimers have been proposed to bind to different locations and also to adopt different conformations (49): one nonstalk dimer of protein L7/L12 is in a folded conformation on the subunit body, while the second dimer is in an extended conformation in the subunit stalk. Other studies (cross-linking data and cryoelectron microscopic localization) also suggest multiple locations for L7/ L12 in the large subunit (18,44,65). The ribosomal stalk is highly flexible, and various conformations of the stalk are thought to occur in response to elongation factor binding and GTP hydrolysis (8).…”

The puzzling lateral flexible stalk of the ribosome

“…RMN experiments on full length P2 made by us have also revealed a high flexibility of the molecule that impeded its 3D determination using this technique (unpublished results). Cross-linking of the extremity of these proteins to several and distant proteins in the ribosome also suggested an important mobility of this structure (Dey et al, 1998).…”