Assembly of L3-L6-ribosomal RNA complex in Bacillus stearothermophilus

Protein synthesis in a cell is carried out by the ribosome. The ribosome, in bacteria, contains more that 4500 nucleotides and 53 proteins and its assembly is known to be a highly cooperative and directed process. The molecular details of the assembly of the ribosome, and other ribonuclear proteins complexes like it are yet not fully understood. However, in order to investigate the problem of assembly, it is important to study a small system which assembles in a few steps. Each step of assembly can then be studied in molecular detail.; The structure of the ribosome has to allow for the many motions that the ribosome goes through during protein synthesis, as well as cyclical interactions with different protein factors. The factor binding site in the large subunit of the ribosome, in particular, known to be mobile, since it interacts with 4 different protein factors during protein synthesis. All four protein factors have been shown to contact the hairpin loop, the Sarcin-Ricin Loop (SRL) which forms a critical part of the binding site for Elongation Factor - G. The crystal structures of the 50S ribosome subunit (Harms et al., 2001, Ban et al., 1999, Schuwirth et al., 2005) show that three ribosomal proteins L3, L6 and L14 bind close to the Sarcin-Ricin Domain (SR domain - helices 94-97) in 23S rRNA. The 12-nucleotide sequence at terminal loop of helix 95 forms the highly conserved SRL (Leffers et al., 1988, Endo et al., 1982). Consequently, it is possible the binding of these three proteins may influence the assembly and conformation of this region, such that the SRL is oriented correctly in the functional ribosome.; Previous studies (Uchiumi et al., 1999) done with Eco proteins showed that both L3 and L6 bind a 156mer rRNA that contains helices h94-97 of the SRdomain of the 23S rRNA. The existence of this rRNA fragment allows the study of the assembly of the small L3-L6-srl156 complex and the conformational changes, if any, induced by the binding of the proteins in the RNA fragment.; Our studies of the renaturation conditions required for L3 and L6 binding to srl156 (a 156nt rRNA fragment containing helices 94-97 from Bacillus stearothermophilus) suggest that L3 initiates rRNA reorganisation or collapse before it can bind srl156, while L6 does not and probably binds to a pre-formed structure. L14 does not appear to bind srl156 strongly enough to be detected by filter-binding assays. Studies also show that L6 binds the loop at the end of H97 strongly (Ka∼109M-1 at 0.1M KCl) using its N-terminal RNA binding site. Using rRNA deletions, we also show that interactions of L6 C-terminal domain with SRL, contributes very little to L6 binding to the full 156mer. Thus, L6 binds the ribosome by essentially binding only the 2730 loop. The weak interaction of the L6 C-terminal rRNA binding site with SRL, offers a site of variable interaction with the SRL and the possibility of modulating its interaction with the elongation factors during the ribosome cycle.; Our experiments also show that while cooperativity is present in the binding of L3 and L6 to srl156, it does not arise in the interactions between L6 and the SRL. Analysis of cooperativity using RNA deletions and insertion mutants show that though cooperativity is not affected by deleting the SRL, it is reduced by a factor of 10 when the orientation of L6 with respect to the rest of the rRNA is distorted. Consequently, it appears that electrostatic interactions play a significant role in the assembly of this complex.