The goal of the proposed research is to understand the assembly of the large ribosomal subunit. We will accomplish this by studying a series of sub-ribosomal particles which arise as a result of mutant or absent assembly cofactors and particles which result from treatment with antibiotics. For instance a single point mutation in the E. coli protein DbpA leads to accumulation of a 45S particle alongside the native 50S particle, and deletion of either SrmB or CsdA results in accumulation of a 40S particle. Additionally, Lincosamides, ketolides and streptogramin B antibiotics have each been shown to specifically inhibit assembly of the large subunit in E. coli. We will identify the composition of these sub-ribosomal particles, and investigate whether they occur as precursors or as a distinct assembly pathway. Liquid chromatography/mass spectrometry of a sample containing the complete trypsin digest of both isotopically labeled and unlabeled ribosomes will generate a pair of peaks for each peptide;one for the unlabeled version and one for the labeled version. If one includes an unlabeled sub-ribosomal particle and a labeled standard subunit of known composition, then the unlabeled fraction of the combined peak areas will give the relative amount of a ribosomal protein in the unknown compared to the standard. In this way, a protein inventory including relative levels can be obtained for each sub-ribosomal particle. One can extend this method to in vivo studies, pulsing an isotopic label into the cell culture and harvesting ribosomes after a variable amount of time to gather kinetic information about incorporation of proteins into the ribosome. A properly functioning ribosome is crucial to cell viability, and has even been implicated as a checkpoint in the cell cycle. Understanding these sub-ribosomal particles will help us to understand the interaction between ribosomes and the checkpoint, along with conditions which might cause the checkpoint to be ignored, leading to uncontrolled cell proliferation. Secondly, a better understanding of antibiotic mechanisms on a molecular level has implications for the clinical use of antibiotics and understanding the onset of resistance in bacteria.
