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

Background: The sequence-specific, hairpin-independent termination signal f or the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules. The upstream module includes the conserved sequen ce and the downstream one is U-rich. Results: Elongation complexes of T7 RNA polymerase paused 2 bp before reach ing 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 th e termination site. Only at higher concentrations of NTP (500 mum), the pau se complex proceeded slowly to and became incompetent at the termination si te. At 4 bp or more before the termination site, the unprotected single-str anded 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 f ew bp prior to the RNA release site. The collapse of bubble that was observ ed several base pairs before the termination site and/or the resulting dupl ex might subsequently lead to the paused conformation of T7 elongation comp lexes.