Fig. 3.

Effect of NI modified-DNA on transcription by T7 RNAP. (A) T7 RNAP transcription was performed as described in Methods for 60 min. The unmodified DNA produced full-length RNA (FL) approximately 340 bases in length (lane 1) while the NI-modified template produced two RNA species, approximately 274 and 340 bases in length (lane 2). A 50 base pair DNA step ladder is labeled M. Shorter RNA fragments are indicated by the grey arrows. These may be due to lesion-induced DNA distortions that are propagated upstream and/or elongating RNA polymerases that are backed up behind the blocked ones, releasing these fragments. (B) Time course experiment to determine lesion bypass efficiency. Transcription was performed as described in Methods and over 90 min with aliquots taken at 0, 1, 10, 20, 30, 40, 50, 60 and 90 min. The undamaged template generates increasing amounts of full-length (FL) transcripts (lanes 1–9) while transcription of the NI-damaged template produces a mixture of truncated and full-length RNA (lanes 10–18). Transcription past NI slows the reaction rate, as indicated by the slow increase in the formation of full-length RNA. The intensity of the bands was compared to a DNA loading control of 103 bases in length. A 50 base pair DNA step ladder is shown (M). Lane numbers are denoted at the top of the gel and time points in minutes at the bottom. (C) Time dependence for formation of full-length and truncated transcripts (displayed as percent of total RNA) over 90 minutes.as determined by band intensity. The values represent the average of three separate transcription trials. As shown, at 1 min the percent of truncated RNA (75% of total RNA) is about 2.5 times greater than that of the full-length transcript (~30% of total RNA); this percentage decreases continuously and after 20 minutes, it stabilizes at ~ 10% of the total RNA produced.