RNA polymerase II ternary complexes may become arrested after transcribing to within 10 bases of the end of linear templates

Abstract

In the presence of elongation factor SII, arrested RNA polymerase II ternary complexes cleave 7-17 nucleotides from the 3'-ends of their nascent RNAs. It has been shown that transcription of linear templates generates apparent run-off RNAs, which are nevertheless truncated upon incubation with SII. By using high resolution gels, we demonstrate that transcription of blunt or 3'-overhung templates with RNA polymerase II generates two populations of ternary complexes. The first class pauses 5-10 bases prior to the end of the template strand. These complexes respond to SII by cleaving approximately 9-17 nucleotide RNAs from their 3'-ends and therefore may be termed arrested. A second class of complexes, which fail to respond to SII, transcribe to within 3 bases of the end of the template strand. These complexes appear to have run off the template since they have released their nascent RNAs. Run-off transcription occurs on all types of templates, but it is the predominant reaction on DNAs with 5'-overhung ends. Thus, RNA polymerase II ternary complexes that retain 5-10 bases of contact with the template strand downstream of the catalytic site become arrested. Further reduction of downstream template contacts can lead to termination. We also show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates.