1. Gene Expression Eukaryotic Gene Transcription 9/18/08 Thomas Ryan, Ph
Gene Expression Eukaryotic Gene Transcription 9/18/08 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics

2. Eukaryotic RNA Polymerases
Three DNA dependent RNA polymerases:
RNA Pol I, II, and III
All 3 are big, multimeric proteins ( kD)
All have 2 large subunits with sequences similar to  and ' in E. coli RNA polymerase, so catalytic site may be conserved
All interact with general transcription factors-GTFs
RNA Pol II is most sensitive to -amanitin

3. RNA Polymerase II Inhibitor a-Amanitin
“The Destroying Angel” - Amanita phalloides
pol II >> pol III >>> pol I
Bicyclic octapeptide
Blocks elongation

4. Yeast RNA Polymerase II Subunits

5. Transcription Factors
The three RNA polymerases (I, II and III) interact with their promoters via protein:protein and protein:DNA interactions
These proteins or transcription factors (TFs) recognize and initiate transcription at specific promoter sequences
Some transcription factors (TFIIIA and TFIIIC for RNA polymerase III) bind to specific recognition sequences within the coding region

6. Helix-Turn-Helix Motif

7. Zinc-Finger Motif: C2H2 Class

8. Basic Region-Leucine Zipper Motif: bZIP

9. bZIP Transcription Factor

10. General Transcription Factors (GTFs)
GTFs position RNAPs at transcription initiation sites, forming the preinitiation complex (PIC)
Transcription-initiation complex = RNAP + general transcription factors (GTF) bound to promoter region
Many of the GTFs that associate with RNAP II initiate transcription from TATA box-containing promoters have been identified
TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
TFII = “transcription factor RNAP II”

11. General Transcription Factors
TFIID is largest and consists of a TATA-box binding protein (TBP) and 8-10 TBP-associated factors (TAFIIs)
TBP is a “universal transcription factor” – associates with promoters of all three RNAPs, and promoters with and without a TATA box
Binding of TAFIIs extend the interactions of TFIID
TFIID has two roles:
foundation for the transcriptional PIC complex
Prevents nucleosome stabilization in the promoter region (antagonist to H1)

12. TBP is used by all 3 RNA polymerases
TBP is a subunit of an important GTF for each of the 3 RNA polymerases:
TBP or TFIID for Pol II
SL1 for Pol I
TFIIIB for Pol III
It does NOT always bind to TATA boxes; promoters for RNA Pol I and Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still used.
The GTFs that contain TBP may serve as positioning factors for their respective polymerases.

13. Yeast TATA Binding Protein: TBP
TBP binds in the minor groove of DNA
TBP binding bends the DNA

14. Promoters for RNAP I
RNA Pol I transcribes genes for the large rRNA precursor
There are hundreds of similar copies of this gene in each genome
RNA Pol I promoters (called class I) have two components:
Upstream control element: –156 to -107
Core element: –45 to +20
Two different transcription factors bind these sequences co-operatively: SL1 and UBF (TBP is a component of SL1)

16. Promoters for RNAP III
Transcribes various small RNAs: 5S rRNA, tRNA precursors, U6 snRNA, etc
The 5S rRNA and tRNA genes have class III promoters
U6 snRNA and 7SL RNA gene have promoters that resemble RNAP II promoters
The 5S rRNA promoters are entirely within the coding region of the gene
The tRNA promoters contain two elements
The other promoters for U6 snRNA etc appear similar to RNAP II promoters (have TATA boxes) but -amanitin test indicates these are transcribed by RNAP III.

17. PIC Assembly for RNA Pol III Genes
TBP
TBP
TBP
TBP

18. RNA Polymerase II General Transcription Factors
Factor Subunits Function
TFIID - TBP 1 Recognize core promoter (TATA), Recruit TFIIB
TFIID - TAFs 12 Recognize core promoter (non-TATA), Pos and Neg regulatory functions, HAT activity
TFIIA 3 Stabilize TFIID and promoter binding
TFIIB 1 Recruit RNA Pol II / TFIIF, Start site selection
TFIIF 2 Assist RNA Pol II to bind promoter
RNA Pol II 12 Enzymatic synthesis of RNA, Recruit TFIIE
TFIIE 2 Recruit TFIIH, Modulate TFIIH helicase, ATPase, and kinase activities
TFIIH 9 Promoter melting using helicase, Promoter clearance via CTD phosphorylation
Modified from Roeder, R.G., Trends in Biochem. Sci. 21:

19. TBP-associated Factors (TAFs or TAFIIs)
Important for helping TBP to bind to promoters that lack TATA boxes.
There are different TAFs in different cells!
In vivo these factors are associated with additional proteins forming a larger complex of about 50 polypeptides.
It is hypothesized that this high MW complex may preassemble and interact with promoters in a single step.

20. Eukaryotic RNA Pol II Transcription
Formation of the PIC
TFIID binds TATA box via TBP subunit
TFIIA facilitates and stabilizes binding of TFIID complex

21. Eukaryotic RNA Pol II Transcription
Formation of the PIC
TFIIB binds to TFIID
TFIIB is a monomeric protein
C terminal domain contacts DNA and TBP
N terminal domain extends towards start site
Proximity of certain promoter- and enhancer-transcription factors (important for developmental regulation- their activation domain binds directly to TFIIB)
TFIIH close as well at this point, but not yet bound

22. Eukaryotic RNA Pol II Transcription
Formation of the PIC
TFIIF binds to RNAPII (preformed complex) – directs RNAPII to promoter
Binding of TFIIE to TFIIF/RNAPII complex and already positioned TFIIB helps positioning the RNAPII over start site:
Two large subunits of RNAP II interact with promoter DNA: CTD tail (unphsphorylated form) of RNAPII is in direct contact with TFIID
TFIIE is DNA-dependent ATPase- probably necessary for generating the energy for transcription

23. Eukaryotic RNA Pol II Transcription
Formation of the PIC
Binding of RNAP II/TFIIF/TFIIE to promoter activates TFIIH
TFIIH contains nine subunits
It has helicase activity- unwinds DNA downstream from the initiator site in the presence of ATP necessary for promoter clearance
It has protein kinase activity- phosphorylation of CTD tail of RNAPII
Phosphorylation detaches RNAPII from TFIID
Beginning of transcription by RNAPII

24. Carboxyl-Terminal Domain (CTD Tail)
Stretch of 7 amino acids that is repeated multiple times (26-52 times):
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
Critical for viability
CTD tail becomes phosphorylated on ser and some tyr residues as the RNAP transcribes away from the promoter

25. RNA Pol II Promoters Consist of two parts: Core promoter:
- TATA box (position at ~ -30)
- initiator (on the transcription start site)
2. Proximal Promoter Elements (can be upstream, downstream or internal)
Upstream
Element
TATA
Initiator
Downstream
Element

26. Eukaryotic TATA Box TATA motif is usually located at position -25
Consensus
Sequence

27. Eukaryotic Promoter Regions
Initiator

28. RNA Polymerase II Promoter Consensus Sequences
of Transcription Factor Binding Sites

29. Modular Factor Binding Sites
Proximal Control Elements of Genes
Modular Factor Binding Sites

30. Coordinate Regulation Via Response Elements
Multiple genes are transcribed in response to different cues: for example, heat shock, hormone levels, developmental events, phorbol esters, heavy metals, metabolite concentrations, etc.
Similarly responsive genes will have a DNA sequence located in cis to the gene called a response element.
These response elements are binding sites for transacting factors that are activated in response to the environmental cue.
The location of these elements relative to the start site of transcription is not conserved between genes: eg. a cis element that leads to transcription in response to a hormone may be located at –300 in one gene and –175 in another

31. Response Elements: Coordinate Regulation
Metallothionein Gene Promoter

32. Coordinate Regulation by Hormones/Steroid Receptors
Adapted from Molecular Biology of the Cell, 4th Edition

33. Steroid Receptors
Upon binding to the hormone cortisol, the cytoplasmic glucocorticoid receptor displaces an inhibitory protein (Hsp90) and moves to the nucleus where is can interact with glucocorticoid response elements (GREs) in the DNA affecting gene transcription.
Adapted from Molecular Biology of the Cell, 4th Edition

34. Activation of Transacting Factors

35. Control of Cellular Differentiation By TFs

36. Enhancers Control elements that stimulate transcription
Bind multiple different transcription factors
Transcription factors that recognize enhancer = activators or enhancer binding proteins
Activators interact with general transcription factors
Negative enhancer is a silencer

37. Enhancers
Stimulate expression of genes over long disances (up to 50kb)
Occur upstream, downstream, in introns or in exons
Orientation independent
May be cell-type specific

38. Enhancers: Action at a Distance
Activation of transcription initiation in eukaryotes by recruitment of the eukaryotic RNA polymerase II holoenzyme complex (100 protein subunits).

39. Insulators / Boundary Elements
Boundary elements block encroachment of heterochromatin from neighboring loci. They also stop the unregulated enhancement or activation of neighboring genes outside of their chromosomal domain.

40. Hypomethylation
(Active)
Vs.
Hypermethylation
(Silenced)

41. Silencing: Histone Deacetylation HDAC
Some repressors recruit histone deacetylase, which removes acetyl groups from histones resulting in gene silencing

42. Histone Acetylation and Deacetylation
Histone acetyl transferases
HAT
HDAC
Histone deacetylases

43. Histone Code
Histone tails are postranslationally modified by acetylation, methylation, phosphorylation and ubiquitination. These modifications have a profound effect on gene activity. The specific set of modifications is termed the “histone code” .

44. Chromatin Remodeling
Remodeling complexes allow access of replication and transcription factors to the DNA.
Remodeling requires ATP

45. Gene Activation By Chromatin Remodeling
Transcription Factor Access

46. HATs open chromatin Nucleosome remodelers Transcription Factor Coactivators

47. Assembly of preinitiation complex on open chromatin

48. Chromatin remodeling of diploid somatic cell nucleus in the egg cytoplasm reprograms the nucleus to recapitulate development.