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  Coupled RNA polymerase II transcription and 3' end formation with yeast whole−cell extracts

Mariconti, L., Loll, B., Schlinkmann, K., Wengi, A., Meinhart, A., & Dichtl, B. (2010). Coupled RNA polymerase II transcription and 3' end formation with yeast whole−cell extracts. RNA-A Publication of the RNA Society, 16(11), 2205-2217. doi:10.1261/rna.2172510.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-5BFE-4 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-5BFF-2
Genre: Journal Article
Alternative Title : Coupled RNA polymerase II transcription and 3' end formation with yeast whole−cell extracts

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 Creators:
Mariconti, Luisa, Author
Loll, Bernhard1, Author              
Schlinkmann, Karola, Author
Wengi, Agnieszka, Author
Meinhart, Anton1, Author              
Dichtl, Bernhard, Author
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: transcription; 3' end formation; termination; yeast, extracts; G-less cassette
 Abstract: 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.

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Language(s): eng - English
 Dates: 2010-03-122010-07-282010-09-012010-09-01
 Publication Status: Published in print
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: RNA-A Publication of the RNA Society
  Other : RNA-Publ. RNA Soc.
Source Genre: Journal
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Publ. Info: New York, NY : Cambridge University Press
Pages: - Volume / Issue: 16 (11) Sequence Number: - Start / End Page: 2205 - 2217 Identifier: ISSN: 1355-8382
CoNE: https://pure.mpg.de/cone/journals/resource/954925343776