2. Transcription in Eukaryotes
⢠Eukaryotic RNA polymerases, unlike their
bacterial counterparts, are incapable of binding
by themselves to their respective promoters
⢠Eukaryotic RNA polymerases rely on proteins
called transcription factors to show them the
way
⢠Two classes: general transcription factors and
gene-specific transcription factors (activators)
3. ⢠General transcription factors combine
with RNA polymerase to form a
preinitiation complex
⢠The assembly of preinitiation
complexes involving polymerase II
is quite complex
5. Structure and Function of TFIID
TFIID contains several subunits
â TATA-box binding protein (TBP)
⢠Highly evolutionarily conserved
⢠Binds to the minor groove of the TATA box
âSaddle-shaped TBP lines up with DNA
âUnderside of the saddle forces open the
minor groove
âThe TATA box is bent into 80° curve
â TBP-associated factors (TAFs) specific for class II
6. TBP [TFIID] function
⢠Binds TATA - main sequence recognition
event during
Binds a variety of different TATA-like sequences
â A slow binding reaction
â minor groove contact
â binds as monomer
⢠Affinity of TBP for TATA contributes to
promoter strength
⢠Binds also several other polypeptides
â activators (Sp1, Tax1, E1A)
â TAFs (dTAF110, dTAF40)
â GTFs (TFIIB, TFIIA)
â inhibitors
⢠TBP = universal TF involved in all three pol
syst.
â TBP i SL1, TFIID, TFIIIB
DNA
Other factors
N
7. TBP versus TFIID
⢠Subunit-structure
â TFIID = TBP + multiple TAFs
â mammalian TFIID: 750 kDa (II), 300 kDa (III) and 200 kDa (I)
â TBP only a small core in the TFIID complex
⢠human 38 kDa, yeast 27 kDa, Arabidopsis 22 kDa
â TBP = N-term divergent domain + C-term. conserved domain
⢠C-term domain 180aa
⢠Carries all essential functions
⢠N-term domain divergent
TAFs
TBP
N
9. TBPs effect on DNA
⢠DNA-structure is distorted upon TBP binding
â DNA severely bended, unwinded and distorted
â DNA shaped by TBP´s ď˘-sheet
â The intercalating Phe-residues contributes to kink
⢠Effect?
â Upstream and downstream elements brought closes together
â incompatible with nucleosome structure
.. but this way
Not like this
10. A Two-Step Mechanism of TBP
Binding to DNA
⢠First step
â Full-length TBPWT first binds
to TATA box to form an
unbent TBP-TATA box
complex.
⢠Second step
â Then, this unbent complex
slowly forms the bent TBP-
TATA box complex.
â TFIIB can directly recognize
the unbent and/or bent
TBP-TATA-complexes to form
the bent TBP-TATA box
complex.
11. 11-11
The Versatility of TBP
⢠Genetic studies have demonstrated TBP
mutant cell extracts are deficient in:
â Transcription of class II genes
â Transcription of class I and III genes
⢠TBP is a universal transcription factor required
by all three classes of genes
⢠Required in transcription of at least some
genes of Archaea, single-celled organisms
lacking nuclei