Open AccessTranscriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.
Author(s) -
Michael Woontner,
Paul A. Wade,
J Bonner,
Judith A. Jaehning
Publication year - 1991
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.11.9.4555
Subject(s) - saccharomyces cerevisiae , biology , upstream activating sequence , transcription (linguistics) , activator (genetics) , dna , transcription factor , general transcription factor , microbiology and biotechnology , rna polymerase ii , rna polymerase , yeast , biochemistry , rna , promoter , gene expression , gene , enhancer , linguistics , philosophy
We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.