How do transcription errors affect cell physiology and what level of the errors can cells tolerate? Progress in answering these questions has been hampered by lack of a reliable selection system for mutations affecting transcription fidelity. We employed S. cerevisiae RNA polymerase II (Pol II) for a combined genetic and biochemical analyses of transcription fidelity control. We selected and characterized a series of novel mutations in Pol II rendering yeast sensitive to 6-azauracil, a drug that unbalances the intracellular NTP pool. Mutation E1103G of the catalytic Rpb1 subunit of Pol II was synthetic lethal with deletion of DST1 (transcript cleavage factor TFIIS) and RPB9 (subunit of Pol II) genes implicated in the RNA proofreading, which implicated glutamic acid 1103 in transcription fidelity control. This conclusion was reinforced by an observed several-fold increase in transcription errors in the cells expressing rpb1-E1103G allele. We analyzed the mutant Pol II in vitro to confirm that the observed cellular phenotypes of the mutations were caused by error-prone transcription rather than alterations in cell physiology or global gene expression. Rpb1-E1103G decreased fidelity of NTP incorporation at least seven-fold, and in a number of the elongation complexes tested over 10-fold indicating that the mutation severely damaged a pre-incorporation fidelity check. The mutant polymerase also exhibited a higher chance for error propagation even in the presence of TFIIS by increasing the rate of the mis-match extension with the next cognate NTP. We proposed that synthetic lethality of rpb1-E1103G and DST1 deletion was caused by an error burst resulting from simultaneous elimination of pre- and post-incorporation fidelity controls based on the cooperation of Pol II and TFIIS. Our results provided the first evidence that transcription error rate is under a strict control in the cell and maintenance of transcription fidelity may be essential for cell viability.
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