Overextended RNA : DNA hybrid as a negative regulator of RNA polymerase IIprocessivity

An eight nucleotide RNA:DNA hybrid at the 3' end of the transcript is requi red for the stability of the elongation complex (EC) of RNA polymerase II. A non-template DNA strand is not needed for the stability of the EC, which contains this minimal hybrid. Here, we apply a recently developed method fo r promoter-independent assembly of functional EC of RNA polymerase II from synthetic RNA and DNA oligonucleotides to study the minimal composition of the nucleic acid array required for stability of the complex with RNA longe r than eight nucleotides. We found that upon RNA extension beyond 14-16 nt in the course of transcription, non-template DNA becomes essential for main taining a stable EC. Our data suggest that the overextended RNA:DNA hybrid formed in the absence the non-template DNA acts as a negative regulator of EC stability. The dissociation of the EC correlates with the backsliding of the polymerase along the overextended hybrid. The dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the EC and of RNA polymerase II processivity.