Abstract 1158 A Pause U C: Transcriptional Pausing on U39C tRNA Phenylalanine Mutant

In mitochondria, RNA Polymerase (POLRMT) carries out the phenomena of transcriptional pausing. I aim to elucidate the role of transcriptional pausing in mitochondrial tRNA sequences and its implications for tRNA modification and mitochondrial diseases. Identifying pause sites for POLRMT permits in-depth studies of POLRMT mechanisms and interactions with external factors. There have been several structural studies that show tRNA modification by MRPP1/2, a methyltransferase protein complex, promotes stability of an active tRNA confirmation. However, certain mutations on tRNA templates preclude these modifications, leading to mitochondrial diseases such as Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). My objective is to identify pause sites on these tRNA templates and investigate how they relate to tRNA modification by MRPP1/2. I hypothesize the time interval created by transcriptional pauses allows for tRNA modification, and mutations on the templates prevent these pauses, resulting in structure malformities and mitochondrial disease. By reconstituting transcription in vitro, I have located a significant pause site on tRNA phenylalanine templates preceding nucleotide U39 addition. Notably, this pausing is absent during the transcription of tRNA Phenylalanine on U39C mutants. Alongside my undergraduate mentees, I purified MRPP1/2, conducted dimethyl sulfate (DMS) probing and methylation by MRPP1/2 experiments. I reverse transcribed the tRNA products to create radioactively labeled complementary DNA and analyzed the products on acrylamide gels. Preliminary findings suggest that these pauses allow for tRNA modifications, and mutations on the templates may prevent these pauses, resulting in structural malformities and mitochondrial diseases. Further investigations utilizing selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) will provide insights into the structural differences between the unmutated and U39C mutated templates and the relationship between MRPP1/2 methyltransferase activity and POLRMT pauses. This project was made possible by: National Institutes of Health/National Institute of General Medical Sciences, Burroughs Wellcome Fund CASI, UCSD institutional support, and Yinan Wang Memorial Chancellor's Endowed Junior Faculty Fellowship to T. V. M., and National Institutes of Health Molecular Biophysics Training Grant to Jubilee Cabellon.