UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.

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