Regulating Ribosome Function Via The Ribosome Exit Tunnel

The large subunit of the ribosome contains the site at which peptide bonds are formed in the process of translation. Another striking feature of the large subunit is the exit tunnel. This feature begins at the site of peptide bond formation traversing 100 angstroms before opening to the cytosolic environment on the opposite side of the large subunit. It has been known for some time that the ribosome exit tunnel is the site of action for MLS antibiotics, one such example being erythromycin. More recently the exit tunnel has been shown to be involved in sensing and regulating the egress of newly synthesized peptides. As the exact mechanisms by which either macrolides such as erythromycin or nascent peptides inhibit ribosome function is not known, understanding how both of these regulatory activities are accomplished remains an important challenge in understanding ribosome structure and function.;Through mutational analysis and the use of translational reporters, I have obtained results which show that, contrary to previously proposed models, the extended loops of these two proteins are not required for ribosomal function, cell survival, or SecM-mediated translational arrest. I also present work showing that the SecM and Crb(CmlA) pausing peptides respond differentially to mutations in L4 and L22. In addition, I have established that the presence of erythromycin inhibits the pausing activity of the SecM peptide. This inhibition takes place even when the pause motif is located well beyond the 6 to 8 amino acid length normally associated with erythromycin inhibition and is dependant on the drug binding. In addition I present evidence that the ability of erythromycin to inhibit SecM pausing occurs translationally and is not attributed to the ribosomes being assembled in the presence of the drug.