Genomic data has led to the realization that nearly one third of the bacterial genes discovered have unknown functions. Promising drug targets are no doubt among the conserved and essential genes, but drug development awaits an understanding of the basic physiological roles of the cryptic bacterial proteins they encode. The pressing need for novel antibiotics mandates investigation of these essential bacterial genes of unknown function. For a subset of such genes, it is becoming increasingly apparent that they may play critical roles in ribosome assembly or function in bacteria. One such essential gene, encoding the putative GTPase YihA in Escherichia coli has been highlighted as a target of particular importance for study (Galperin M.Y. and E.V. Koonin. (2004). 'Conserved hypothetical' proteins: prioritization of targets for experimental study. Nucleic Acids Res 32(18): 5452-63.). To study yihA's function, a strain with yihA under P_BAD control was generated and used to examine the impact of the loss of the gene product on ribosome assembly. Depletion of the YihA protein was found to cause accumulation of 30S and 50S subunits at the expense of 70S ribosomes, and the formation of long filamentous cells apparently incapable of dividing. Chemical genetic interactions were identified through chemical synthetic lethality screening with antibiotics which target the ribosomal A -site, and antibiotics which affect the bacterial cell wall. Finally, investigation of the importance of several patches of basic residues of YihA through mutagenesis and in vivo complementation shows the protein to be tolerant of mutation to non-catalytic residues. The inability of mutants with changes to amino acids important for nucleotide binding to complement represents the strongest evidence to date that the YihA protein relies on GTP binding and hydrolysis as part of its essential function in vivo. The findings of this study support a role for YihA in the poorly understood process of ribosome assembly in vivo, and represent important progress towards realization of this essential process as a source of novel antibiotic targets. / Thesis / Master of Science (MS)