Project Summary In the cell, the RNA made by RNA polymerase (RNAP) folds into its functional three-dimensional shape while it is being synthesized by RNAP. The kinetics of RNA synthesis, which determine the RNA folding outcome, are influenced by myriad factors, such as intracellular temperature, pH, concentrations of small molecules and proteins in the cell, and the exact sequence of DNA being transcribed into RNA. Transcription is not a continuous process: RNA synthesis by RNAP is interrupted by sequence-dependent pauses, during which RNAP remains bound to the nucleic acids without active nucleotide addition occurring. These pauses create windows of time for regulation of transcription to occur. Our research program will address the mechanisms of pausing at the atomic level and the contribution of pausing to co-transcriptional events, such as folding of RNA, in bacteria and human mitochondria. The first direction of the program aims to develop tools for capturing and visualizing RNA folding intermediates during transcription and to understand the effect of pH on the kinetics of RNA synthesis by RNAP, and thus the RNA folding pathway. The resulting tools will be of broad interest to the RNA community because they can be applied to follow folding of other biologically important RNAs. A second direction will apply those tools to map the differences in co-transcriptional RNA folding of “healthy” and mutated human mitochondrial transfer RNAs (mt-tRNA), thus providing the structural basis for disease-causing mt-tRNA mutations. We will assess the contribution of mitochondrial RNAP (mtRNAP) pausing to the differential folding of unmutated vs. disease-variant mt-tRNA. Additionally, we will test another hypothesized function of mtRNAP pausing: coupling of transcription of mitochondrial DNA (mtDNA) to its replication, which is critical for maintaining enough mtDNA copies for production of protein components of the oxidative phosphorylation machinery. Finally, a third research direction will address how the balance between transcription of mtDNA and its packaging is achieved to cater to the ever-changing cellular needs for energy. The completion of the proposed research will be transformative to the understanding of basic principles governing gene expression, the molecular mechanisms of diseases linked to mtDNA, and to the applications of RNA-based tools in synthetic biology.