Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail. Mutagenic compounds are a potent source of human disease. By inducing genetic instability, they can accelerate the evolution of human cancers or lead to the development of genetically inherited diseases. Here, we show that in addition to genetic mutations, mutagens are also a powerful source of transcription errors. These errors arise in dividing and nondividing cells alike, affect every class of transcripts inside cells, and, in certain cases, greatly exceed the number of mutations that arise in the genome. In addition, we reveal the kinetics of transcription errors in response to mutagen exposure and find that DNA repair is required to mitigate transcriptional mutagenesis after exposure. Together, these observations have far-reaching consequences for our understanding of mutagenesis in human aging and disease, and suggest that the impact of DNA damage on human physiology has been greatly underestimated. The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a Tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found faster tRNA binding of the near-cognate tRNA after initial unsuccessful tRNA sampling and a strong negative correlation between the ambient concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to an altered ribosomal configuration that assists in rejecting subsequent incorrect tRNA interactions.