Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+and NADH, using NAD+and NADH as non canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to

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... 50% NAD+and ~40% NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at and upstream of the transcription start site and, in yeast and human cells, by intracellular NAD+and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+and NADH levels and adjusting transcriptional outputs accordingly.