The objective of this research program is to understand the SsrA (tmRNA) system of E. coli, to probe its roles in ribosome rescue, protein tagging, and other cellular processes, and to determine how bacterial and phage proteins bearing ssrA tags or other degradation signals are recognized and degraded by bacterial proteases. We will probe the mRNA and/or protein determinants that induce SsrA tagging, study the biological function of full-length protein tagging, and determine which macromolecular factors associate with SsrA RNA during different parts of the tmRNA cycle. We will also study the structure, function, and substrate-binding specificity of ClpXP, the major protease that degrades ssrA-tagged proteins, and SspB, a modulatory factor that collaborates with ClpXP to enhance degradation of ssrA-tagged proteins. Repressors and other important regulatory factors are frequent targets of ClpXP degradation. Understanding SsrA function, ribosome rescue, and protein degradation are key goals of basic research in molecular and structural biology, with potential applications in medicine, biotechnology, and the design of novel proteins and regulatory circuits. For example, the SsrA system is required for the infectivity of bacterial pathogens and allows bacteria to withstand higher doses of antibiotics that inhibit protein synthesis. Analysis of the sites of SsrA tagging reveals locations of ribosome distress, providing a unique glimpse of the molecular events that hinder or impede protein biosynthesis. Such information could permit improved expression of recombinant proteins. ClpX serves both as the regulatory subunit of the ClpXP protease and as an AAA+ family disassembly chaperone. A detailed knowledge of ClpX-substrate recognition could allow the design of enzymes with altered specificity for use as tools in discovery research. ClpX, ClpXP, and SspB also serve as models in which to understand molecular mechanisms that have been conserved from bacteria to humans and adapted by all cells in processes ranging from protein degradation to membrane fusion. ? ?
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