Structural studies of the 70S ribosome and its associated factors

The ribosome is a large ribonucleoprotein complex that is responsible for carrying out messenger directed protein synthesis in the cell. A variety of different translation factors are recruited to the ribosome at various stages of the translation cycle to aid the ribosome in protein synthesis.;LepA is a highly conserved elongation factor that promotes the back translocation of the tRNAs on the ribosome during the elongation cycle. We determined the crystal structure of LepA from Escherichia coli to 2.8 A. The high degree of sequence identity between LepA and EF-G is reflected in the structural similarity between the individual homologous domains of LepA and EF-G. However, the orientation of domains III and V in LepA differs from their orientations in EF-G. LepA also contains a C-terminal domain (CTD) not found in EF-G that has a novel protein fold. We derived a homology model for LepA binding to the 70S in which the electrostatically positive CTD of LepA is placed in the direct vicinity of the A-site of the large ribosomal subunit suggesting a possible interaction between the CTD and the back translocated tRNA or 23S rRNA.;EFP is a universally conserved and essential protein found in all eubacteria that stimulates the formation of the first peptide bond. We solved the crystal structure of EFP bound to the 70S ribosome along with an initiator tRNAi and a fragment of mRNA to 3.3 A resolution which reveals that EFP binds next to the P-site tRNA between the P- and E-tRNA binding sites. EFP spans both ribosomal subunits with its N-terminal domain positioned next to the aminoacyl acceptor stem of the P-site tRNA and its C-terminal domain positioned next to the anticodon stem loop of the P-site tRNA. The binding of EFP to the ribosome causes a large conformational change of the L1 stalk that moves the ribosomal protein L1 into the E-site to interact with domain II of EFP. The essential function of EFP in the cell appears to be assuring the proper positioning of the fMet-tRNAifMet for the first step of protein synthesis by preventing its inappropriate forward or back translocation.