Approximately 40 percent of the total energy production of E. coli is consumed to synthesize the large number of ribosomal components, 3 RNAs totaling over 4000 nucleotides and greater than 50 proteins. In bacteria, the rates of cell growth and ribosome biosynthesis are intimately related and directly correlated. Thus, for cell viability, the synthesis of ribosomal components and their subsequent assembly must be coordinated. These observations suggest that an accurately assembled and functional ribosome is one of the most important cellular organelles and that defects in ribosome assembly would be fatal. Although, years of research have been dedicated to studying ribosomal components and ribosome function, the pathway(s) and dynamics of ribosome assembly remain largely uncharacterized despite general importance to cell physiology. This proposal describes three approaches to study the mechanism, pathway and dynamics of ribosome assembly. The goal of this proposal is to understand what factors are involved in and critical for E. coli 30S ribosomal subunit assembly. First, in vitro reconstitution of 30S subunits using a complete set of recombinant small subunit ribosomal proteins will be used to identify l6S rRNA nucleotides and small subunit ribosomal proteins involved in 30S subunit assembly. Second, a combination of solution chemical probing and small subunit ribosomal protein-directed hydroxyl radical probing will be used to map 16S rRNA folding during different stages of 30S subunit formation. Lastly, in vitro reconstitution and standard biochemical approaches will be used to purify and characterize cellular (extra-ribosomal) factors involved in ribosome assembly. Preliminary results suggest that all three specific aims will add to our understanding of ribosome assembly. Insights gained in these studies will allow us to pursue future experiments studying ribosome biogenesis in vivo. In addition, these studies may identify targets for regulation of bacterial cell growth and perhaps lead to the production of novel antimicrobials.
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