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.
Small intergenic repeat elements called REPs, and perhaps other hairpin-shaped mRNA structures, can regulate gene expression when in the DNA downstream from E. coli genes. However they have to be located within the first 15 bases after the stop codon of a gene to exert their effects. We explore the extent and mechanism of this new regulatory role for trans-translation further.