1. Transcription in eukaryotes
•RNA Polymerases
•Promoters
•General Transcription Factors

2. Model of yeast RNA polII with RNA-DNA hybrid in the active site

3. Eukaryotes have 3 different RNA
polymerases

5. Amanita phalloides

6. a-Amanitin

8. Sensitivity or RNA Pols to a-amanitin

9. Structure of RNA Polymerases
•all 3 are large multimeric proteins ( kDa)
•all have two large subunits with sequence similarity
to b and b’ of E. coli. Therefore, catalytic sites of
three RNA Pol I, II and III may be conserved.

10. Epitope tagging to Identify RNA Pol II subunits

11. Subunit structure of yeast RNA polymerase II

12. RNA Polymerase II Structure Summary
•Yeast Pol II enzyme consists of 12 different poly-
peptides (RPB1 - 12).
•Core subunits are RPB 1, 2 and 3.
•RPB1 and 2 are orthologues of E. coli RNA Pol b’ and b.
•RPB1 has DNA binding site and has carboxy-terminal
domain (CTD). CTD has repeating heptad PTSPSYS -
hydrophilic sequence.
•5 of 7 aa’s of heptad have ‘-OH’ groups that can be
phosphorylated

13. RNA Polymerase II Structure Summary Continued
•RPB2 (b ortholgue) is at or near active site.
•RPB3/11 may function as orthologue of a-dimer of
prokaryotic RNA Pol
•RPB5, 6, 8, 10 and 12 are shared between RNA Pol I,
II, and III
•RPB4 and 9 are non-essential RNA Pol II subunits

14. Structure of Prokaryotic and Eukaryotic RNA Pols

15. Eukaryotic Promoters
•3 classes of eukaryotic promoters: I, II and III -
transcribed RNA Pol I, II, and III respectively.
•Class I promoters not well conserved in sequence
amongst different species but general architecture is:
consists of a core element and an upstream promoter
element (UPE).
•Some Class III promoters have promoter
sequences wholly within the gene. Another type of Class III
promoter resembles Class II promoters.
•Class II promoters have up to five conserved elements.

16. First consensus sequence from lining up
several eukaryotic promoters: TATA box

17. Generic class II promoter
TFIIB
recognition
Downstream
Promoter
element

18. Transcription Factors
•Eukaryotic RNA Pols cannot bind to promoters on
their own (unlike prokaryotic RNA Pols).
•Transcription factors are required for eukaryotic
RNA Pol binding.
•General transcription factors allow binding of RNA Pol
binding to promoters and a basal level of transcription.
•Gene-specific factors stimulate transcription further
(or repress it) and allow fine regulatory control.

19. Six general transcription factors: TFIIA, B, D,
F, E and H initiate transcription.
•Gel shift assays showed that TFIID, A and B can form
a complex independently on DNA (i.e. without RNA
RNA Pol)
•TFIIF binding to DAB complex is dependent on RNA
Pol. Therefore, RNA Pol and TFIIF are needed
together to join preinitiation complex.
•Binding of TFIID to the TATA box occurs first; This
event is required for binding of all of the other TFs.
•In vitro, TFIIA is not critical for preinitiation complex
formation.

20. Model for DABPolF complex formation

23. TFIID •TFIID consists of a TATA-box binding protein (TBP)
and eight TBP-associated factors (TAFIIs).
•TBP is well conserved between different organisms.
•C-terminal 180 amino acids of TBP is sufficient
for binding to the TATA box.
•TBP interacts with the minor groove of the TATA box
-unusual.
•TBP also functions with Pol I and III. It is a universal
transcription factor.

24. TBP-Associated Factors - TAFIIs
•TAFIIs attach to TBP and extend binding of TFIID
beyond TATA box in some promoters
• TAFIIs can bind initiator and downstream elements;
TAFIIs help initiate transcription from promoters
initiators and DPEs
•Specifically, TAFII150 and TAFII250 form a ternary
complex with TBP and bind to the initiator and DPE
-shown by crosslinking and footprinting studies
•In certain TATA-less promoters, TAFII250 and
TAFII150 bind to initiator or DPE and recruit rest of
TFIID to promoter

25. Drosphila TFIID assembled in vitro

28. Enhancers and Silencers
•Class II promoters have cis-acting DNA elements
that influence transcription but are not part of the
promoter: enhancers and silencers
•Trans-acting factors - proteins - act in conjunction
with cis-elements.
•Enhancers stimulate transcription while silencers
inhibit transcription. Certain elements can act as
silencers or enhancers depending on the trans-acting
factors present.
•Enhancers and silencers are position and orientation-
independent.

29. Transcription activators:
DNA binding motif
-Zinc containing modules
-Homoedomains
-bZIP and bHLH domains
Activation domain
-Acidic
-Glutamine rich
-Proline rich
Diversity of interaction with other transcription factors - semester course in molecular biology on this topic alone

30. Model for transcriptional activation

31. Model for initiation, promoter clearance,
and elongation

32. Summary Picture

33. Eukaryotic transcription termination
by RNA Pol II
•Mechanism of termination remains unclear.
•Involves at least two elements: a polyadenlyation
signal and a downstream element (DSE) involved in
Pol II pausing.