The research in the laboratory focuses on understanding the fundamental mechanisms of translation and action of ribosome-targeting antibiotics. One of the underappreciated aspects of translation that has transpired from our studies is its context specific nature. We learned that the ability of the ribosome to decode genetic information rapidly and efficiently is significantly influenced by the nature of its tRNA and mRNA substrates and/or properties of the polypeptide being made. Specific sequences have been identified as difficult to polymerize but, more than likely, there are many other that also present an obstacle for the translating ribosome. Why some sequences are challenging for translation and what mechanisms exist in the cell to help the ribosome overcome such problems are questions that have remained unanswered. Several conserved auxiliary translation factors (auxTFs) encoded in the bacterial genomes are thought to assist the ribosome in synthesizing proteins during fast growth or under stress, likely facilitating translation of the problematic sequence motifs. However, our knowledge of the nature of the sequences whose translation is facilitated by the auxTFs, about the molecular mechanisms of their action and of their physiological significance is limited at best. Answering these vexing questions is the goal of the proposal. We will use crosslinking-based ribosome profiling (XLRibo-seq) to map the genomic sites where auxTFs are recruited to the ribosome. Guided by these genome-wide data, we will select the most revealing gene models to dissect in vitro the precise nature of the problematic sequence motifs and elucidate the molecular mechanisms of the auxTF action. The inferred mechanisms will be validated in vivo and the physiological role of the auxTFs will be deduced by following the expression of specifically designed reporters under suitable growth conditions or using the appropriate mutants. The overall experimental approach will be first optimized using, as a model system, translation termination factors RF1 and RF2, and then applied to four most intriguing auxTFs (LepA, EttA, BirA and HflX) whose specificity, activity and physiological role remain enigmatic despite being investigated previously by other techniques. The approach could be extended to several other known or suspected auxTFs and even to the main translation factors whose activity could be conceivably modulated by the context of the translated sequence. The innovation of the proposal stems from the development of new methodologies (XLRibo-seq) with a broad application to studies of basic mechanisms of translation, and from revealing the functions and molecular mechanisms of action of conserved proteins playing important roles in expression of genetic information. The experiments build upon the long-term interests, previous findings, and expertise of our research group. The project will provide new training and teaching opportunities and facilitate the outreach ...