BACKGROUND: The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in transcription regulation. Guanine-rich sequences form structures called guanine quadruplexes that result from non-canonical base pairing between guanine residues. These stable guanine quadruplex structures are prevalent in gene promoters in nuclear DNA and are known to be associated with promoter proximal pausing of some genes. However, the transcriptional impact of guanine quadruplexes that form in nascent RNA is poorly understood. RESULTS: We examined mitochondrial RNA polymerase (POLRMT) pausing patterns in primary human skin fibroblast cells using the precision nuclear run-on assay and uncovered over 400 pause sites on the mitochondrial genome. We identified that these pauses frequently occur following guanine-rich sequences where quadruplexes form. Using an in vitro primer extension assay, we show that quadruplexes formed in nascent RNA act as mediators of POLRMT pausing, and in cell-based assays their stabilization disrupts POLRMT transcription. Cells exposed to a guanine-quadruplex stabilizing agent (RHPS4) had diminished mitochondrial gene expression and significantly lowered cellular respiration within 24 h. The resulting ATP stress was sufficient to reduce active transport in renal epithelia. CONCLUSIONS: Our findings connect RNA guanine quadruplex-mediated pausing with the regulation of POLRMT transcription and mitochondrial function. We demonstrate that tuning of quadruplex dynamics in nascent RNA, rather than template DNA upstream of the polymerase, is sufficient to regulate mitochondrial gene expression.