Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

التفاصيل البيبلوغرافية
العنوان: Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts
المؤلفون: Anton Meinhart, Bernhard Dichtl, Luisa Mariconti, Agnieszka Wengi, Karola M. Schlinkmann, Bernhard Loll
المساهمون: University of Zurich, Dichtl, B
المصدر: RNA-A Publication of the RNA Society
بيانات النشر: Cold Spring Harbor Laboratory Press, 2010.
سنة النشر: 2010
مصطلحات موضوعية: Cell Extracts, mRNA Cleavage and Polyadenylation Factors, Saccharomyces cerevisiae Proteins, biology, General transcription factor, Transcription, Genetic, Termination factor, RNA polymerase II, RNA, Fungal, Saccharomyces cerevisiae, Molecular biology, 10124 Institute of Molecular Life Sciences, Article, Cell biology, 1312 Molecular Biology, biology.protein, 570 Life sciences, Transcription factor II F, Transcription factor II E, RNA Polymerase II, Transcription factor II D, Molecular Biology, RNA polymerase II holoenzyme, Transcription factor II B, Protein Binding
الوصف: RNA polymerase II (RNAP II) transcription and pre-mRNA 3′ end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3′ end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 3′ end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m7G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 3′ end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 5′-3′ exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.
وصف الملف: 2205.full.pdf - application/pdf; application/pdf
اللغة: English
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c0917e6785d38a09e5b55c2168f19376
https://europepmc.org/articles/PMC2957059/
Rights: OPEN
رقم الانضمام: edsair.doi.dedup.....c0917e6785d38a09e5b55c2168f19376
قاعدة البيانات: OpenAIRE