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<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

<p>Using the model organism <em>Escherichia coli</em>, we discuss herein two novel antimicrobial targets: namely, the protein YjeE and the process of ribosome assembly.</p> <p>YjeE is essential for viability and widely conserved amongst bacterial pathogens and has no human homologue. We searched for a small molecule probe of the function of YjeE to help circumvent the inadequate genetic tools that are available for studying this protein. Sensitive methods for detecting ligand binding were optimized; however, this effort yielded no inhibitors. A second approach to studying the function of YjeE was the development of a reporter using a promoter that is directly upstream of <em>yjeE </em>in <em>E. coli</em>. The activity of this promoter was tested in the presence of small molecules of known function and in diverse gene deletion backgrounds. YjeE found to be linked to the inhibition of DNA and protein translation as well as central metabolism and respiration. These interactions prompted experiments that revealed YjeE to be dispensable under anaerobic conditions.</p> <p>Many antibiotics target ribosomal protein synthesis; however, no current antibiotics target the process of ribosome biogenesis. In order to identify new biogenesis factors, the non-essential fraction of the <em>E. coli </em>genome was screened for deletions that gave rise to cold-sensitive growth. We found that genes associated with ribosome function were the most represented cold sensitive factors amongst the genes of known function. We identified and present here two new putative ribosome biogenesis factors, <em>prfC</em> and <em>ychF</em>, which had phenotypes associated with ribosome assembly defects.</p> / Doctor of Philosophy (PhD)

Yes / The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation. / This work was supported by grants from the BBSRC [H007202/1] and Wellcome Trust [097820/Z/11/A].