Mechanism of transcription and related gene expression processes in bacteria and human mitochondria

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Project Summary The synthesis of RNA by RNA polymerase (RNAP) and folding of that RNA into its biologically functional three- dimensional shape go hand in hand. The kinetics of RNA synthesis determine the folding outcome, and 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 the polymerase 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 at the atomic level the pausing mechanisms and contribution of pausing to co-transcriptional events, such as folding of RNA. 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. The second direction will apply those tools to map co-transcriptional RNA folding differences in “healthy” and mutated human mitochondrial transfer RNAs (mt-tRNA), thus providing the structural basis for disease-causing mt-tRNA mutations. Additionally, we will assess the contribution of mitochondrial RNAP pausing to the differential folding of unmutated and disease-variant mt-tRNA, thus expanding the arsenal of regulatory roles transcriptional pausing plays in this key organelle. Finally, the third research direction will address how the balance between transcription of mitochondrial DNA 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 the basic principles of gene expression, as well as to the applications in synthetic biology and to the molecular mechanisms of diseases linked to mitochondrial DNA.