PROJECT SUMMARY Proteins carrying out DNA repair, replication and transcription, processes essential for the stable transmission and expression of genomic information, share the same track – the DNA – and require delicate coordination to achieve their function. Often, regulation of these processes occurs at the level of targeting or well-timed recruitment, but also catalysis and even turnover of the factor in question. We will address these three principal modalities of regulation in the context of bacterial responses to stress signals. Our questions and hypotheses are grouped into two large areas, (1) regulation of the general stress response mediated by the promoter specificity subunit RpoS; this is exemplary of a general stress response mechanism that affect transcription initiation, and (2) specific responses to DNA damage such as subpathways of nucleotide excision repair; these are exemplary of how the DNA damage response interfaces with transcription elongation, termination as well as replication. The principal questions that will be addressed by my research plan are: A. How are activating and inhibitory regulatory inputs integrated to tune the RpoS core pathway and globally reprogram transcription in response to an adverse environment? How does the ClpXP adaptor RssB and stress-specific ClpXP anti-adaptors tune the proteolysis of RpoS in a stress-specific manner? B. What are the mechanisms for preferential recruitment of the NER machinery to specific lesions, or to the template strand, directly read by RNA polymerase? How is the availability of early NER factors regulated by proteolysis in the context of DNA damage and transcription-coupled DNA repair? How and when do transcription-repair coupling factors collaborate with key players in the SOS response to promote mutagenesis? Can we leverage these mechanisms towards developing novel anti-evolution drugs for combination therapies against infection with diverse pathogens? Previous work has already allowed us to build a critical collection of reagents and expertise in the structural biology and biochemistry of transcription-coupled repair and RpoS biology. This gives us now an excellent entry point for a mechanistic dissection of the processes listed above and the development of novel antimicrobial strategies. The significance of our work is thus not only fundamental and conceptual in nature, but has immediate applications in controlling the worldwide public health crisis of antimicrobial resistance, particularly in the age of the COVID-19 pandemic.