Improved methods for expression and purification of Saccharomyces cerevisiae TFIIF and TFIIH; identification of a functional Escherichia coli promoter and internal translation initiation within the N-terminal coding region of the TFIIF TFG1 subunit.
The basal RNA polymerase II (RNAPII) transcription machinery is composed of RNAPII and the general transcription factors (TF) TATA binding protein (TBP), TFIIB, TFIIE, TFIIF and TFIIH. Due to the powerful genetic and molecular approaches that can be utilized, the budding yeast Saccharomyces cerevisiae has proven to be an invaluable model system for studies of the mechanisms of RNAPII transcription. Complementary biochemical studies of the S. cerevisiae basal transcription machinery, however, have been hampered by difficulties in the purification of TFIIF and TFIIH, most notably due to the severe toxicity of the TFIIF Tfg1 subunit in Escherichia coli and the complexity of the purification scheme for native TFIIH. Here, we report the elimination of TFG1-associated toxicity in E. coli, the identification and removal of a functional E. coli promoter and internal translation initiation within the N-terminal coding region of TFG1, and the efficient production and two-step purification of recombinant TFIIF complexes. We also report conditions for the efficient two-step tandem affinity purification (TAP) of holo-TFIIH, core TFIIH and TFIIK complexes from yeast whole cell extracts.
Yang
Chen
C
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214-3000, USA.
Khaperskyy
Denys A
DA
Hou
Min
M
Ponticelli
Alfred S
AS
D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000502:physiology; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D051758:Transcription Factor TFIIH / Q000235:genetics* / Q000302:isolation & purification; D019473:Transcription Factors, TFII / Q000235:genetics* / Q000302:isolation & purification
D021122:Protein Subunits; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; C056604:transcription factor TFIIF