Structural Analysis of the 50S Ribosomal Stalk

Abstract

Protein biosynthesis represents a dynamic process that takes place on the ribosome and is driven by translation factors. Some of these factors are GTP binding proteins. They possess a limited inherent GTPase activity that is stimulated by interactions with the ribosome in a region located on the large ribosomal subunit (GTPase associated region).This site comprises several 23S rRNA elements (L10/L11 rRNA binding region and sarcin-ricin loop) and r-proteins, such as L6, L11, L14, and the L7/L12 stalk. The latter corresponds to an extended feature of the 50S ribosomal subunit, encompassing multiple copies of protein L12 that are linked to the ribosomal RNA via L10. Numerous lines of evidence indicated that L12 is essential for both translation factor binding and stimulation of their GTPase activities. Functionally, L12 can be divided into an N-terminal domain (NTD) responsible for dimerization and interaction with L10, a C-terminal domain (CTD) necessary for factor-related functions, and an intervening flexible hinge.Crystallographic studies of 50S subunits and 70S ribosomes hitherto failed to disclose the structure of the L7/L12 stalk, most probably due to the high mobility of the L12 hinge region. Thus, a complex anticipated to exhibit less flexibility was designed. It encompassed L10 and the NTD of L12 from the hyperthermophilic bacterium Thermotoga maritima. In the three crystal structures obtained, L10 displayed a globular NTD connected by a flexible loop to a long C-terminal α-helix. The latter displayed different orientations relative to the L10 NTD in different crystal forms and harbored three consecutive binding sites for the L12 NTD dimers. Such a 1:6 (L10:L12) stoichiometry was unexpected, as a 1:4 ratio was well established in E. coli. The L12 NTDs formed dimers that fitted to a mode of dimerization reported for the protein in isolation, both in solution (Bocharov et al. 2004;Moens et al. 2005) and in crystalline environment (Wahl et al. 2000a). In the crystal structure of isolated T. maritima L12, the hinge region of one protomer exhibited an α-helical shape, folded onto the L12 NTDs of the dimer, while in tmaL10:(L12 NTD) 6 , the hinge was found replaced by the C-terminal α-helix of L10. Thus, it is likely that in complex with L10, the L12 hinges are flexible and unstructured, in agreement with several studies of this protein in solution.In addition to obtaining the structure of tmaL10:(L12 NTD) 6 , attempts to solve the crystal structure of the full-length L10:L12 complex were also undertaken. While the 1 Summary crystallization of the complex from the hyperthermophilic bacterium Aquifex aeolicus proved to be unsuccessful, the corresponding complex from Thermotoga maritima yielded crystals that diffracted to 3.5 Å. The structure could be solved by molecular replacement using the tmaL10:(L12 NTD) 6 complex as a search model. No electron density could be detected for the L12 hinges and CTDs, consistent with a degradation of L12 during crystallization, as revealed by SDS-PAGE analysis of dissolved cryst...