Transcription in human mitochondria by mitochondrial RNA polymerase (POLRMT) is coupled to mitochondrial DNA (mtDNA) replication, and the mechanism of how transcription switches to replication is not fully understood. Transcriptional pausing, which is predicted to occur upstream of this switch, could play an important regulatory role. My project aims to fill the gap in knowledge about the mechanism of transcriptional pausing by POLRMT, its potential regulatory roles on human mtDNA and its effects on mtDNA replication. To accomplish this goal, I have successfully reconstituted transcription in vitro on DNA sequences encoding human mtDNA promoters and the downstream conserved sequence block (CSB) regions. In these reactions, I observed paused RNA species corresponding to the positions identified by previous in vivo sequencing of pause sites. Going forward, I will elucidate the mechanism of POLRMT pausing at these sites by in vitro transcription assays, use single-particle cryo-EM to identify conformational changes in POLRMT in the paused state, and employ 6-methylisoxanthopterin (6-MI), a fluorescent nucleotide analog, as a probe of the translocation state of the nucleic acids in the paused transcription complex. I will compare these paused POLRMT attributes to T7 RNA polymerase, a structurally similar RNAP that does not pause during transcription.