Sequence-specific termination by T7 RNA polymerase requires formation of paused conformation prior to the point of RNA release

Abstract

Background: The sequence-specific, hairpin-independent termination signal for the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules. The upstream module includes the conserved sequence and the downstream one is U-rich. Results: Elongation complexes of T7 RNA polymerase paused 2 bp before reaching the termination site at a 500 mum concentration of NTP. At 5-50 mum NTP, however, they paused and terminated there or resumed elongation beyond the termination site. Only at higher concentrations of NTP (500 mum), the pause complex proceeded slowly to and became incompetent at the termination site. At 4 bp or more before the termination site, the unprotected single-stranded region of transcription bubble shrank at the trailing edge to 4-5 bp from approximate to 10 bp, resulting from duplex formation of the conserved sequence. The pause and bubble collapse were not observed with an inactive mutant of the termination signal. Conclusion: Sequence-specific termination requires the slow elongation mode of paused conformation, working only at high concentrations of NTP for a few bp prior to the RNA release site. The collapse of bubble that was observed several base pairs before the termination site and/or the resulting duplex might subsequently lead to the paused conformation of T7 elongation complexes.