ELUCIDATING THE FUNCTION OF ASSEMBLY FACTORS IN THE MATURATION OF THE BACTERIAL LARGE RIBOSOMAL SUBUNIT

Abstract

Antibiotic resistance in bacteria is becoming a major threat to public health. Many of the antibiotics used today in the clinic target the process of protein synthesis performed by the ribosome. Recent prospects for blocking ribosome function are increasingly focusing on preventing the assembly of bacterial ribosomes. A number of ribosome assembly factors are emerging as attractive targets for novel antibiotics that work in new ways. YphC and YsxC are essential GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit; however, their roles in this process are still uncharacterized. To explore their function, we biochemically and structurally characterized the 45SYphC and 44.5SYsxC precursor particles accumulated from strains depleted of YphC and YsxC, respectively. Quantitative mass spectrometry analysis and 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in maturation of functional sites of the 50S subunit. We also observed that YphC and YsxC bind specifically to the two immature particles. In addition, we characterized the structure of the 50S subunits in complex with the RbgA protein. The preliminary 3D structure shows that the RbgA protein binds to the P site of the 50S subunit and displaces h69. There are also missing densities in the structure for h68 and the uL16 ribosomal protein. We expect that the atomic resolution structure of the 50S.RbgA complex will reveal the function and molecular mechanisms of this assembly factor. The deep structural understanding of protein synthesis process done by the ribosome led to the optimization of over a hundred antibiotics that are currently used in thev clinic. In the same manner, work described in this thesis provides novel insights into understanding the maturation of the large ribosomal subunit, and is paving the way to use the bacterial ribosome biogenesis pathway as a target for the development of new antimicrobials.