Regulation of translation optimizes both the speed and fidelity of protein synthesis. Many quality control steps ensure that the correct amount of properly folded protein is produced. When problems arise during translation, ribosome rescue systems are recruited to resolve stalled ribosomes and restore protein production. Ribosome stalling can result in RNase-mediated degradation of the coding region of the mRNA. One such RNase activity, termed A-site mRNA cleavage, results in ribosomes stalled on truncated mRNAs. These ribosomes recruit the tmRNA ribosome rescue system. tmRNA acts as a quality control molecule by facilitating stalled ribosome resolution. Furthermore, translation of a tmRNA-encoded ORF appends a peptide tag to the incomplete protein, targeting it for degradation. Together, these processes resolve problems during protein synthesis and recycle resources for optimal growth.
This body of work utilizes both molecular and biochemical methods to investigate the causes and consequences of ribosome pausing in Escherichia coli. Chapter 1 presents a general introduction outlining the translation cycle, the tmRNA ribosome rescue system, ribosome pausing, and RNase activities. A ribosome pause with little tmRNA activity is presented in Chapter 2. The secretion monitor (SecM) system utilizes ribosome stalling for gene regulation, but prevents most tmRNA activity by filling the A-site with a proline tRNA. Kinetics of peptidyl-tRNA turnover during ribosome stalling is measured in Chapter 3. This includes evidence of the existence of a hypothesized tmRNA-independent system for resolving stalled ribosomes. In Chapter 4, the requirement for mRNA degradation/cleavage of mRNA prior to tmRNA activity during ribosome pausing is explored. Surprisingly, the results demonstrate that A-site mRNA cleavage is not necessary for tmRNA activity on stalled ribosomes. A description of the heat shock activation of a known A-site nuclease (YoeB) acting on stalled ribosome complexes is presented in Chapter 5. Two separate pathways that induce A-site cleavage, one RNase II-dependent and one RNase II-independent (YoeB-mediated) are considered. The final chapter summarizes the results of this dissertation and presents a revised model for ribosome pausing and A-site mRNA cleavage.
|Commitee:||Foltz, Kathleen, Low, David, Orias, Eduardo|
|School:||University of California, Santa Barbara|
|Department:||Molecular, Cellular & Developmental Biology|
|School Location:||United States -- California|
|Source:||DAI-B 72/03, Dissertation Abstracts International|
|Keywords:||Ribosomes, Translation, mRNA cleavage|
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