DNA lesions can severely compromise genome stability and lead to cell death if unrepaired. RNA polymerase (RNAP) is known to serve as a sensor of DNA damage and to attract DNA repair factors to the damaged template sites. Here, we systematically investigated the ability of Escherichia coli RNAP to transcribe DNA templates containing various types of DNA lesions, and analyzed their effects on transcription fidelity. We showed that transcription is strongly inhibited on templates containing cyclobutane thymine dimers, 1,N6-ethenoadenine and abasic sites, while 8-oxoguanine and thymine glycol have mild effects on transcription efficiency. Similarly to many polymerases, E. coli RNAP follows the "A" rule during nucleotide insertion opposite abasic sites and bulky lesions, and can also incorporate and efficiently extend an adenine nucleotide opposite 8-oxoguanine. Mutations in RNAP regions around the templating nucleotide decrease the efficiency of translesion synthesis, likely by altering the RNAP-template contacts in the active site. Thus, DNA lesions can lead to distinct outcomes in transcription, depending on the severity of the damage and contacts of the damaged template with the active site of RNAP. (C) 2019 Elsevier Inc. All rights reserved.