Chromatographic Separation of the three forms of RNA Polymerase II.
α-amanitin
Function of the three enzymes.
Subunit structure.
Yeast Pol II.
Epitope-tagging
Epitope-tagged purification of RNA polymerase II from yeast.
Comparison of yeast RNA
polymerase II with E. coli core
and holoenzyme
Model of Yeast RNA Polymerase
II
Crystal structure of yeast RNA
polymerase II
structure
structure
structure
structure
structure
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structure
In vitro transcription assays
Fractionation of Pol II GTFs
from HeLa cells
DABF Pol II complex
Structure of TBP bound to DNA.
TAFs.
TAF comparison.
TAF activity
In vivo Dependence on TAFs.
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The Drosophila tudor gene has
two promoters one uses TBP and
the other TRF-1
TFIIA
• Yeast 32kD, 13.5kD
• Human and fly 30kD, 30kD, and 13kD
• Binds to IID and stabilizes protein-DNA
interaction
TFIIB
• Yeast 26kD
• Humans 35kD
• Single subunit allows polymerase to
associate with the DAB complex
TFIIF
• Yeast 105kD, 55kD, 30kD
• Humans 70kD, 30kD
• Binds to RNA polymerase and interacts
with TFIIB in the DAB complex
TFIIE
• Yeast 66kD and 43kD
• Humans 56kD and 34kD
• Required for in vitro transcription activity
TFIIH
• Yeast 9 subunits
• Humans 8 subunits
• Contains a kinase activity (cdk7) that can
phosphorylate the CTD of the largest
subunit of RNA polymerase II
• This phosphorylation is required for the
enzyme to elongate transcription
Preinitiation complex.
Initiation to elongation.
IIS
IIS
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