1. Promoters and Enhancers

2. Introduction Basic of transcription
Proteins/
transcriptional
factors
Figure 24.1
Basic of transcription
Typical gene transcribed by RNA polymerase II
(Transcriptional ) Basal Factors may induce transcription or cause the enhancement or
repression of transcription. Enhancers may be distant from the promoter (from bp to kb).
Basal Transcription Apparatus

3. Eukaryotic RNA Polymerases Consist of Many Subunits
RNA polymerase I synthesizes rRNA (18, 5.8 and 28S rRNA) in the nucleolus.
RNA polymerase II synthesizes mRNA (some snRNA and miRNA) in the nucleoplasm.
RNA polymerase III synthesizes small RNAs (tRNA, 5S rRNA and other snRNA) in the nucleoplasm.

4. In plants
--RNA polymerase IV synthesizes siRNA
--RNA polymerase V synthesizes siRNA-directed heterochromatin
In bacteria
---DNA-dependent RNA polymerase synthesizes mRNA and ncRNA.

5. Some subunits are common to all three RNA polymerases.
All eukaryotic RNA polymerases have ∼12 subunits and are aggregates of >500 kD. (nucleotide pair~0.660 kD)
Some subunits are common to all three RNA polymerases.
The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer.
-Typical RNA polymerase isolated from yeast (S. cerevisiase) ( and  subunits)
- ’ subunit: CTD – carboxy-terminal domain, which consists in multiple repeats of 7 amino acids, unique and important of regulation (tyrosine, serine and threonine residues)
-Some subunits are common to all three polymerases.
-Polymerases can be distinguished by their sensitivity to α-amanitin, Pol-II and III are sensitive. Pol I is insensitive
Figure 24.2

7. Promoter Elements Are Defined by Mutations and Footprinting (DNA-protein interaction?)
Promoters are defined by their ability to cause transcription of an attached sequence in an appropriate test system in vitro or in vivo. ( enhancers, or silencers
Identification
Promoter elements can be defined by testing deletion mutants in either the frog oocyte, transfection, transgenic or in vitro system.
Characterization
Footprinting determines where transcription factors bind and protect the promoter from DNAse digestion.
Figure 24.3
Identification and characterization of the promoters region

8. Four systems to study promoters
Oocyte system – inject the gene into the nucleus of an X. laevis oocyte. Restricted to the conditions that occur in the oocyte.
Transfection systems – introduce exogenous DNA into a cultured cell. Expression can be assayed. The exogenous gene would not usually be expressed in the cell. Useful to study basic transcription, not so useful for developmental regulation.
Transgenic systems – introduce exogenous DNA into the germline. Expression of the transgene can be followed in any tissue at any stage of development. Problems are with number and location of integration of transgene.
In vitro system – purify all components and control all conditions until faithful initiation is seen. Probably the only way to initially define the role of all components (Reticulocyte system; nuclear fraction)

9. RNA Polymerase I Has a Bipartite Promoter
RNA Pol I transcribes rRNA genes.
The promoter has two regions: the upstream control element (UC-P-E) and the core promoter.
Core promoter: -45 to +20 seq.,
G-C-rich and A-T-rich (Inr-initiator) regions,
Core binding factors -complex formed by 4(?) proteins, which contains TBP-(TATA-AAA- binding protein) and binds to “the core” and positions Poly-I.
UCE: G-C-rich region, -180 to -107
UBF1 binds to GC rich regions of the UCE, and is necessary for high frequency initiation.
UBF1 increases the efficiency of the binding of the core binding factors
UBF1 and TBP complex allows the incorporation of the RNA Poylmerase I binds to the starting-point.
The RNA polymerase I promoter consists of:
--a core promoter
--an upstream control element (UC-P-E)
Figure 24.5

10. RNA polymerase binds to the UBF1-SL1 complex at the core promoter.
The factor UBF1 wraps DNA around a protein structure to bring the core and U(P)CE into proximity.
SL1 includes the factor TBP that is involved in initiation by all three RNA polymerases.
RNA polymerase binds to the UBF1-SL1 complex at the core promoter.
Initiation of transcription by RNA polymerase I requires the formation
of “one complex” composed of the TATA-binding protein (TBP) and
three (?) TBP-associated factors (TAFs) specific for RNA polymerase I.
This complex is also known as SL1. It binds to the core promotion

11. RNA Polymerase III Uses Both Downstream and Upstream Promoters
RNA polymerase III has two (3) types of promoters.
-RNA Pol III transcribes tRNA and small RNA
-Core promoters (boxes)
-Transcriptional Factors (TF) III: general and specifics
*proximal sequence element *
Figure 24.7

12. TFIIIA (a zinc finger protein:DNA-binding protein) and TFIIIC (protein complex) are assembly factors for TFIIIB which can initiate transcription even if TFIIIA and C are removed.
TFIIIA and TFIIIC: assembly factors ; TFIIIB true initiate factor.
TFIIIB: protein complex-3 subunits complex (TBP-BRF-B”)
Poly III binds to TFIIIB complex.

13. TFIIIB Is the Commitment Factor for Pol III Promoters
TFIIIA and TFIIIC bind to the consensus sequences and enable TFIIIB to bind at the startpoint.
TFIIIB has TBP as one subunit and enables RNA polymerase to bind.
Figure 24.9

14. RNA Polymerase II Type promoters for Poly III -upstream elements
-common to Poly II promoters
--TATA (specificity of Poly III)
--ELEMENTS
----PSE (efficiency of the transcript)
----Oct (efficiency of the transcript)
TFIIIA and TFIIIC: assembly factors
TFIIIB true initiator factor and directs the position of the RNA polymerase to the
starting-point.
RNA Polymerase II

15. The TATA box is a common component of RNA polymerase II promoters
It consists of an A-T-rich octamer located ~25 bp upstream of the startpoint.
The DPE is a common component of RNA polymerase II promoters that do not contain a TATA box.
A core promoter for RNA polymerase II includes:
the InR (CA and Py)
either a TATA box or a DPE Y=
Py--pyrimidines--
DPE=
Downstream
Promoter
Element
Figure 24.10

16. TBP Is a Universal Factor
TBP is a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter.
The factor for RNA polymerase II is TFIID, which consists of:
TBP
11 TAF(TBP- associated proteins)s
The total mass is ∼800 kD.
Figure 24.11

17. -TFIID: TATA binding protein (TBP) and TBP- associated proteins (TAF)
RNA Polymerase II
-TFIID: TATA binding protein (TBP) and
TBP- associated proteins (TAF)
-~800 KD protein complex
Eukaryotic polymerases bind specifically to protein factors, not DNA.
The factors that position the RNA polymerase at the transcription start all contain TBP (TATA binding protein).
TBP is important for RNA Pol I, II and III
(Universal Factors)
Transcriptional Factors bind DNA
-minor groove followed by a change in conformation.
-A-T rich regions
- nucleosomes ?
TBP binds in the minor groove, which would
normally face the histone octamer surface.
This may be why promoters do not have
nucleosomes.
-Additional protein complex
in the major groove

18. TBP Binds DNA in an Unusual Way
TBP binds to the TATA box in the minor groove of DNA.
It forms a saddle (supportive structure) around the DNA and bends it by ∼80°.
Some of the TAFs resemble histones and may form a structure resembling a histone octamer.

20. Initiation of Transcription:
RNA polymerase, protein factors, and cis-acting elements on DNA
The sequence of assembly of the factors is important.
1-step: complex formation (TFIID)
2-step: complex binding to DNA (TBP) and TAFs
3-step: TFIIA and B.
4-step: TFIIF together with TFIIB ( and others TFII E and J) assemble the RNA Polymerase to the transcriptional complex.
5-step: initiation of the transcription by the addition of the TFIIF… How?
TFIIF is an ATP-dependent DNA helicase…..melting DNA at initiation
TFIIE and TFIIH will extend and melt the DNA
TFIIH is the only factor with independent enzyme activity. One is an ATP-dependent helicase of both polarities and a kinase activity that can phosphorylate the CTD tail of Poly-II.

21. The Basal Apparatus Assembles at the Promoter
Binding of TFIID to the TATA box is the first step in initiation.
Other transcription factors bind to the complex in a defined order
This extends the length of the protected region on DNA.
When RNA polymerase II binds to the complex, it initiates transcription.
Figure 24.14

22. Initiation Is Followed by Promoter Clearance
TFIIE and TFIIH are required to melt DNA to allow polymerase movement.
Phosphorylation of the CTD may be required for elongation to begin.
Further phosphorylation of the CTD is required at some promoters to end abortive initiation.
Figure 24.17

23. The CTD may coordinate processing of RNA with transcription.
Most factors leave before transcription initiates
The CTD tail is phosphorylated (energy requiring modification) and then RNA Poly II begins to elongate mRNA.
How could this phosphorylation process regulate the initiation of transcription?
The phosphorylated CTD recruits enzymes that modify RNA, such as capping of the newly transcribed mRNA by the capping enzyme (guanylyl transferase), splicing (SCAF proteins) and polyadenylation at the 3’ end.
The CTD
may coordinate
processing of
RNA with
transcription.
P-TEFb (Kinases?)
Figure 24.18

24. P-TEF beta (Kinase?) It is Cyclin-Dependent Kinase It phosphorylates …
the negative regulating factor (DSIF) the negative elongation factor (NELF)

25. RNA Pol II pre-initiation complex (PIC)

26. A Connection between Transcription and Repair
Transcribed genes are preferentially repaired when DNA damage occurs.
The template strand is preferentially repaired.
Mfd in prokaryotes facilitates repair of the template strand. How? “It removes stalled RNA poly”
(Uvr Enzymes complex)
In eukaryotes, TFIIH may be involved in binding the repair complex.
Figure 24.19

27. TFIIH provides the link to a complex of repair enzymes.
Mutations in the XPD component of TFIIH cause human diseases
(Xeroderma Pigmentosa -XP-genes)
Stability of TFIIH
Figure 24.20

28. TFIIH (XPD, XPB, p62, p52, p44, p34 and TTDA)
(CDK7, cyclin H and MAT1)

29. Short Sequence Elements Bind Activators
Activators and Enhancers
Short Sequence Elements Bind Activators
Short conserved sequence elements are dispersed in the region preceding the startpoint.
The upstream elements increase the frequency of initiation.
The factors that bind to stimulate transcription are called activators.

30. What is the advantage of having a number of different sequence elements in the promoter region?
Activators can be mixed and matched in the promoter, but no one activator is essential.
TATA:-30
GC: -90
CAAT: -75
Activators
Repressors

31. Promoter Construction Is Flexible but Context Can Be Important
No individual upstream element is essential for promoter function;
Although one or more elements must be present for efficient initiation.
Some elements are recognized by multiple factors.
The factor that is used at any particular promoter may be determined by the context of the other factors that are bound.
Figure 24.22

32. Enhancers Contain Bidirectional Elements That Assist Initiation
An enhancer activates the nearest promoter to it.
It can be any distance either upstream or downstream of the promoter.
Enhancers can act up to several kb away, either upstream or downstream, in either orientation. Enhancers increase transcription at all nearby promoters.
Enhancers may concentrate factors at the promoter site by looping to bring the enhancer close to the promoter.
Enhancers can be found in gene transcriptional units under special circumstances, such as immunoglobulin genes that must be rearranged prior to gene expression.

33. A UAS (upstream activator sequence) in yeast behaves like an enhancer but works only upstream of the promoter.
Similar sequence elements are found in enhancers and promoters.
Enhancers form complexes of activators that interact directly or not with the promoter.

34. Enhancers Contain the Same Elements That Are Found at Promoters
Enhancers are made of the same short sequence elements that are found in promoters.
The density of sequence components is greater in the enhancer than in the promoter.
Figure 24.24

35. Enhancers can be made to work in trans configuration by:
Enhancers Work by Increasing the Concentration of Activators Near the Promoter
Enhancers usually work only in cis configuration with a target promoter.
Enhancers can be made to work in trans configuration by:
linking the DNA that contains the target promoter to the DNA that contains the enhancer via a
- protein bridge
- catenating the two molecules

36. The principle is that an enhancer works in any situation in which it is constrained to be in the same proximity as the promoter.
Figure 24.25

37. Gene Expression Is Associated with DNA De-methylation
Gene expression is associated with de-methylation Me Me
Isoschizimers can identify methylation state of CpG (5-CG-3’). C is methylated at C5 in the pyrimidine ring.
Genes active in one tissue but inactive in others usually are unmethylated when active.
Treatment with 5-azacytidine (analog of cytidine) can lead to demethylation of C and the induction of gene transcription. However, some genes can be expressed when are heavily methylated.

38. CpG rich islands are regulatory targets
CpG-rich islands occur in the promoters and in the transcribed region of housekeeping genes and are unmethylated.
Activators may not bind at methylated sites and/or Repressors may only bind at methylated sites.
When are the CpG rich islands important?
No TATA and CAAT boxes
1/100bp
~4 to 5/100 bp
APRT: adenine phospho-ribosyl-transferase

39. CpG Islands Are Regulatory Targets
CpG islands surround the promoters of constitutively expressed genes where they are unmethylated.
CpG islands also are found at the promoters of some tissue-regulated genes.

40. There are ∼29,000 CpG islands in the human genome.
Methylation of a CpG island prevents activation of a promoter within it.
Repression is caused by proteins that bind to methylated CpG doublets.

41. Modulation of Regulatory Domain
A domain may have:
an insulator
an LCR
a matrix attachment site
transcription unit(s)
Protection
MAR
-Domains might define the loops of DNA that attach to
the nuclear matrix and allow local reduction of
supercoiling and displacement of histones from
nucleosomes.
-Domains may be an intermediate level in chromosome
architecture.
Figure 29.54
Large size of domains “as regulatory units” must “decondense” chromatin for access by RNA polymerases. This may be one reason why eukaryotic genomes are bigger than prokaryotic genomes.
(MAR: matrix attachment region, LCR: locus control region)

42. Lamin proteins

43. Insulators Block the Actions of Enhancers and Heterochromatin
Insulators are able to block passage of any activating or inactivating effects from:
Enhancers, Silencers, LCRs
Insulators are DNA sequences that prevent the either activation or inactivation of genes transcription.
-prevent the spread of activating or inactivating effects.
-are placed between an enhancer and promoter prevents activation.
-are placed between a gene and heterochromatin prevents inactivation.
Figure 29.42

44. Insulators may provide barriers against the spread of heterochromatin (HP1 protein).
Figure 29.43