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 down-stream 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.
Highlights
From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524
To determine the nature of transcript cleavage in complexes that have reached the end of a linear template, we used the pML20 plasmid, which contains the Ad 2 ML promoter, linearized with one of the following restriction endonucleases: Hindiii, Bgli (+220, 3'-overhung end), Fspi (+213, blunt end), and Pvull (+ 163, blunt end)
The strategy we employed to generate ternary complexes on linear templates was essentially identical to that used on circular templates [17]
Summary
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 truncated upon incubation with SII. We show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates It has been appreciated for some time that a fraction of the transcribing RNA polymerase II ternary complexes may stop transcription after traversing certain sequences, thereby entering a state called arrest The sites that provoke arrest typically encode runs of U within the transcript; other sequence features are usually present as well [1, 2] Arrested polymerases retain their RNAs in ternary complex but cannot continue transcription. The large difference in SII-facilitated cleavage increment between elongation-competent stalled complexes and elongation-incompetent arrested complexes strongly suggests that arrest is accompanied by an internal reorganization of the ternary complex
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
