The ability to properly perform protein synthesis is essential to the survival of cells. This process, called translation, is catalyzed by the ribosome and must be done both at high speed and with high fidelity. Problems inherently occur during translation and must be dealt with by ribosome rescue systems to restore proper protein synthesis. The tmRNA-SmpB ribosome rescue system acts to resolve stalled ribosomes and also adds a peptide tag to incomplete proteins, which targets them for degradation. The tmRNA-SmpB ribosome rescue system and the alternative ribosome rescue system mediated by ArfA work in parallel to resolve problems occurring during protein synthesis, allowing proper translation to continue.

This body of work uses biochemical and molecular techniques to investigate the causes of problematic protein synthesis as well as the systems in place to deal with theses problems and their regulation. Chapter 1 provides an introduction to translation and the translation cycle, the tmRNA-SmpB ribosome rescue system, mutations effecting translation termination, and the alternative ribosome rescue system mediated by ArfA. The effects of mutating the ribosome modifying pseudouridine synthase RluD is presented in chapter 2. This mutation causes an increase in tmRNA-mediated tagging of small ribosomal protein S7, resulting in slow growth. The regulation of the alternative ribosome rescue factor ArfA by tmRNA is discussed in chapter 3. In E. coli, tmRNA acts on the arfA transcript because the transcript contains a stem-loop, which is cleaved by RNase III. This results in ArfA being created off of a non-stop mRNA. Chapter 4 looks at the regulation of ArfA in other proteobacteria and discusses how ArfA of the beta-proteobacteria Neisseria gonorrhoeae is created off of non-stop mRNA due to an intrinsic terminator within the open reading frame rather than by the action of RNases. Chapter 5 summarizes the work presented in this dissertation, examines its importance, and proposes questions about this work that still need to be answered.