Accurate transcription is required for the faithful expression of genetic information. To understand the molecular mechanisms that control the fidelity of transcription, we used novel sequencing technology to provide the first comprehensive analysis of the fidelity of transcription in eukaryotic cells. Our results demonstrate that transcription errors can occur in any gene, at any location, and affect every aspect of protein structure and function. In addition, we show that multiple proteins safeguard the fidelity of transcription and provide evidence suggesting that errors that evade these layers of RNA quality control profoundly affect the physiology of living cells. Together, these observations demonstrate that there is an inherent limit to the faithful expression of the genome and suggest that the impact of mutagenesis on cellular health and fitness is substantially greater than currently appreciated.