hide
Free keywords:
RNA polymerase; RNase; DNA topology; R-loop
Abstract:
The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than ~10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of ~170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides broadblocksQ to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.
