English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

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

MPS-Authors
/persons/resource/persons94115

Loll,  Bernhard
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons94313

Meinhart,  Anton
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002C-5BFE-4
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.