Factor-independent termination of transcription involves a complex interplay between RNA polymerase, DNA, and nascent RNA. While class I termination is reasonably well-characterized, involving formation of a hairpin in the exiting RNA to pull the RNA out of the hybrid (shearing) and/or to drive hyper-forward translocation to shorten the hybrid. In the T7 RNA polymerase model system, class II termination, which involves a relatively short recognition sequence, is more poorly understood, despite ample prior characterization. In this study, we apply a range of techniques to re-characterize class II termination (and pausing) and discover an unprecedented phenomenon in transcription that has been overlooked by traditional studies. RNA-seq characterization of transcription products reveals the expected terminated and full length products, but also identifies a family of new short RNA products, that arise exclusively during termination. Since these products are of similar length to abortive initiation products arising during initiation, prior experiments using gel electrophoresis overlooked their existence. Having observed these products in RNA-seq, differential labeling with fluorescent base analogs confirms their existence in electrophoretic analyses. Mass spectrometry reveals that these products are not degradation products and instead arise from de novo initiation at the termination site. The results indicate that promoter-initiated RNA is released at termination, but the RNA polymerase remains bound to the DNA and can initiate promoter-independent transcription from that location, producing a range of RNA lengths. Finally, we explore structural models that may allow for this unprecedented phenomenon.
Abek et al. (Sun,) studied this question.