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

Mariconti, Luisa, Loll, Bernhard, Schlinkmann, Karola, Wengi, Agnieszka, Meinhart, Anton and Dichtl, Bernhard 2010, Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts, RNA, vol. 16, no. 11, pp. 2205-2217, doi: 10.1261/rna.2172510.

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Title Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts
Author(s) Mariconti, Luisa
Loll, Bernhard
Schlinkmann, Karola
Wengi, Agnieszka
Meinhart, Anton
Dichtl, BernhardORCID iD for Dichtl, Bernhard orcid.org/0000-0001-5514-4982
Journal name RNA
Volume number 16
Issue number 11
Start page 2205
End page 2217
Total pages 13
Publisher Cold Spring Harbor Laboratory Press
Place of publication Woodbury, N.Y.
Publication date 2010-11-16
ISSN 1355-8382
1469-9001
Summary  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 eng
DOI 10.1261/rna.2172510
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2010, Cold Spring Harbor Laboratory Press
Free to Read? Yes
Persistent URL http://hdl.handle.net/10536/DRO/DU:30053077

Document type: Journal Article
Collections: School of Life and Environmental Sciences
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Created: Sun, 23 Jun 2013, 15:48:58 EST by Bernhard Dichtl

Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.