Cryo-electron microscopy (cryo-EM) studies of mammalian mitochondrial and Mycobacterium smegmatis ribosomes
Ribosomes the protein synthesis machinery are large macromolecular complexes. The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation (or ATP generation) for the eukaryotic cell. The mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. The cryo-EM structures of the mitoribosome have yielded the architecture of small subunit of the mitoribosome and existence of the previously eluded E-site within the mitoribosome. The mitoribosomes are susceptible to the antibiotics that targets the bacterial ribosomes because the mitoribosomes are believed be of prokaryotic origin. A docking study of antibiotics in to bacterial and mitochondrial ribosomes have provided rational to the differential affinity of these antibiotic.
Mycobacterium smegmatis is widely used as a model organism to study pathogenic mycobacteria, such as Mycobacterium tuberculosis. About one third of M. smegmatis mRNAs are leaderless, lacking a 5’UTR and therefore also lacking a Shine-Dalgarno sequence. How ribosomes initiate protein synthesis from leaderless mRNAs is unknown. We have obtained a 5 Å resolution cryo- EM map of the M. smegmatis 70S ribosome, with some core regions resolved to 4 Å. Our map reveals two unique features of the mycobacterial ribosome: (i) an altered conformation of the previously identified steeple feature on the large (50S) ribosomal subunit (ii) a novel protein density mass is found below the origin of the rim of the small (30S) ribosomal subunit platform that appears to correspond to an α-helical structure of up to ~33 amino acid residues. While we continue to improve the resolution of our cryo-EM map, we are also using mass spectroscopy to identify the α-helical density. I will summarize the results of these studies that I have been involved in, and will outline my future research plan on the protein synthesis in Plasmodium falciparum ribosome that I would like to pursue as an independent investigator.