PROJECT SUMMARY A novel function for REP family repetitive DNA elements encoded distal and proximal (<16 bp) to protein-coding regions in the E. coli K-12 genome has been recently discovered. These elements signal a partial, transient, and reversible translational stalling mediated through trans-translation resulting in a three-fold modulation of protein and mRNA levels. This work extended the cellular roles for the essential and universal trans-translation process to include gene regulation. This research proposal aims to broadly extend and confirm the initial report, potentially impacting many different areas of bacterial physiology due to (1) a large number and functional diversity of potentially regulated genes, (2) the experimental REP element legacy and a wide distribution of REP elements among both medically threatening and biome healing Enterobaceae, and (3) the likelihood that the signaling elements are not restricted only to REP-encoded RNA stem-loops. The complementary goals of this proposal are (1) to elucidate the biochemical pathways and signaling mechanisms involved experimentally in E. coli K-12 case studies, and (2) to apply bioinformatics to identify, functionally assess, and cluster genes that all have REPS and other RNA structures <16 bp after stop codons in common. Specific Aim 1 will determine the sequence requirements of mRNA stem-loops for the induction of trans-translation. Specific Aim 2 will examine the physiological significance and roles of REP hairpins, and the examination of physiological inducers, including the known UV induction in more detail. Specific Aim 3 will explore the mechanism of initiation of trans-translation by REPs. We will examine the roles of small mRNA interferases and rare stop codons in REP regulation. Specific Aim 4 will determine the distribution of predicted REP-arrested genes in the enteric bacteria and look for functional commonalities, especially their involvement in stress responses.