1. Promoters and Enhancers

2. Ex Biochem c24-promoter enhancer
24.1 Introduction
Significant difference between transcription of eukaryotic and prokaryotic mRNA
Initiation at eukaryotic promoter involves many factors that bind to a variety of cis-acting elements
Eukaryotic RNA polymerase bind around the startpoint, but NOT directly contact the extended upstream region of the promoter
Eukaryotic Promoter 起始區域, 啟動子, 啟動區域: the region containing all these binding sites
Major feature defining promoter for eukaryotic mRNA: location of binding sites for transcription factors
Bacterial promoter: binding site for RNA polymerase in the immediate vicinity of startpoint

3. Ex Biochem c24-promoter enhancer
24.1 Introduction
Transcription factors are needed for initiation, but not required subsequently
In eukaryotes, the transcription factors are principally responsible for recognizing 辨識promoter
Bacterial RNA polymerase recognize promoter
RNA Polymerase II require a large group of transcription factors: basal factors
RNA Pol I and III relatively simple
Basal transcription apparatus: basal factors + RNA polymerase

4. Ex Biochem c24-promoter enhancer
24.1 Introduction
The sequences farther upstream of promoter determine whether the promoter is expressed in all cell types or specifically regulated
Constitutively expressed promoter (for housekeeping genes) have upstream sequence elements recognized by ubiquitous activators
Beta-actin, glucose-6-phosphate dehydrogenase
Promoters expressed only in certain times/places have sequence elements that require activators available only at those times/places

5. Ex Biochem c24-promoter enhancer
Enhancers 強化子, 強化區域
Another type of site involved in initiation 起始
Sequences that stimulate initiation, but located a considerable distance from startpoint
Often targets for tissue-specific or temporal regulation
Components of enhancer resemble those of promoter
Consist of a variety of modular elements
Proteins bound at enhancer interact with proteins bound at promoter
Eukaryotic transcription usually under positive regulation
Less by repression regulation

6. Ex Biochem c24-promoter enhancer
24.1 Introduction
Figure 24.1

7. 24.2 Eukaryotic RNA Polymerases Consist of Many Subunits
Ex Biochem c24-promoter enhancer
24.2 Eukaryotic RNA Polymerases Consist of Many Subunits
RNA polymerase I synthesizes rRNA in nucleolus.
RNA polymerase II synthesizes mRNA in nucleoplasm.
RNA polymerase III synthesizes small RNAs in the nucleoplasm.
All eukaryotic RNA polymerases have ∼12 subunits and are aggregates of >500 kD.
Largest subunit in RNA Pol II has carboxy-terminal domain (CTD), which consists of multiple repeats of a consensus sequence of 7 AA (YSPTSPS)
Some subunits are common to all three RNA polymerases.
RNA polymerase in mitochondria and chloroplasts
Smaller
Resemble bacterial RNA polymerase

8. Ex Biochem c24-promoter enhancer
Figure 24.2

9. Ex Biochem c24-promoter enhancer

10. 24.3 Promoter Elements Are Defined by Mutations and Footprinting
Ex Biochem c24-promoter enhancer
24.3 Promoter Elements Are Defined by Mutations and Footprinting
Promoters are defined by their ability to cause transcription of an attached sequence in an appropriate test system in vitro or in vivo.
Figure 24.3

11. 24.4 RNA Polymerase I Has a Bipartite Promoter
Ex Biochem c24-promoter enhancer
24.4 RNA Polymerase I Has a Bipartite Promoter
The RNA polymerase I promoter consists of:
a core promoter: -45 to +20
an upstream control element (UPE): -180 to -107
Figure 24.5

12. 24.4 RNA Polymerase I Has a Bipartite Promoter
Ex Biochem c24-promoter enhancer
24.4 RNA Polymerase I Has a Bipartite Promoter
Requires 2 ancillary factors
The factor UBF1 wraps DNA around a protein structure to bring the core and UPE into proximity.
UBF: upstream binding factor
SL1 (core-binding factor) includes the factor TBP that is involved in initiation by all three RNA polymerases.
TBP: TATA-binding protein
RNA polymerase binds to the UBF1-SL1 complex at the core promoter.

13. 24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters
Ex Biochem c24-promoter enhancer
24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters
RNA polymerase III has two types of promoters.
Figure 24.7

14. 24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters
Ex Biochem c24-promoter enhancer
24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters
Internal promoters:
have short consensus sequences located within the transcription unit
cause initiation to occur a fixed distance upstream
Upstream promoters contain three short consensus sequences upstream of the startpoint that are bound by transcription factors.
Figure 24.6

15. 24.6 TFIIIB Is the Commitment Factor for Pol III Promoters
Ex Biochem c24-promoter enhancer
24.6 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.
TFIIIA and TFIIIC: assembly factors whose only role is to assist binding of TFIIIB at right location
TFIIIB has TBP as one subunit and enables RNA polymerase to bind.
Figure 24.9

16. Figure 24.08: Type 2 internal promoters use TFIIIC.
Ex Biochem c24-promoter enhancer
Figure 24.08: Type 2 internal promoters use TFIIIC.

17. Ex Biochem c24-promoter enhancer

18. 24.7 The Startpoint for RNA Polymerase II
Ex Biochem c24-promoter enhancer
24.7 The Startpoint for RNA Polymerase II
RNA polymerase II requires general transcription factors (called TFIIX) to initiate transcription.
RNA pol II promoters have a short conserved sequence Py2CAPy5 (the initiator, Inr) at startpoint.
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 a common component of RNA pol II promoters that do not contain a TATA box.
down-stream promoter element,
A core promoter for RNA polymerase II includes: TATA box + Inr, or Inr + DPE

19. Ex Biochem c24-promoter enhancer
Figure 24.10

20. 24.8 TBP Is a Universal Factor
Ex Biochem c24-promoter enhancer
24.8 TBP Is a Universal Factor
TATA-binding protein (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 TAFs (TBP-associated factors)
The total mass is ∼800 kD.
Positioning factors containing TBF and TAFs responsible for identifying all classes of promoters
Figure 24.11

21. 24.9 TBP Binds DNA in an Unusual Way
Ex Biochem c24-promoter enhancer
24.9 TBP Binds DNA in an Unusual Way
TBP binds to the TATA box in the minor groove of DNA.
It forms a saddle around the DNA and bends it by ∼80°.
Some of the TAFs resemble histones and may form a structure resembling a histone octamer.

22. 24.10 The Basal Apparatus 基礎成分 Assembles at the Promoter
Ex Biochem c24-promoter enhancer
24.10 The Basal Apparatus 基礎成分 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
TBP binds to the TATA box in the minor groove of DNA.
Figure 24.14

23. Figure 24.16: TFIIB helps position RNA polymerase II.
Ex Biochem c24-promoter enhancer
Figure 24.16: TFIIB helps position RNA polymerase II.

24. 24.11 Initiation Is Followed by Promoter Clearance
Ex Biochem c24-promoter enhancer
24.11 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.
Figure 24.17

25. Ex Biochem c24-promoter enhancer
Roles of CTD
CTD may be a general focus for connecting other processes with transcription
Directly or indirectly involved in processing RNA after it synthesized by RNA polymerase
Bind to capping enzyme
Bind to SCAFs, then binding to splicing factors
Bind to components of cleavage/polyadenylation apparatus
Figure 24.18

26. Ex Biochem c24-promoter enhancer

27. 24.13 Short Sequence Elements Bind Activators
Ex Biochem c24-promoter enhancer
24.13 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.
Control the efficiency and specificity with which a promoter is recognized
TATA box: -30
CAAT box : -75
GC box: -90

28. Ex Biochem c24-promoter enhancer
Figure 24.21: The ß-globin promoter has three short sequence elements.
Correspond to TATA, CAAT, and GC boxes

29. Ex Biochem c24-promoter enhancer
24.14 Promoter Construction Is Flexible 彈性 but Context 內容Can Be Important
Promoters organized of ‘mix and match’ principle
A variety of elements can contribute to promoter function
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.
Figure 24.22

30. 24.15 Enhancers Contain Bidirectional Elements That Assist Initiation
Ex Biochem c24-promoter enhancer
24.15 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.
Figure 24.23

31. 24.15 Enhancers Contain Bidirectional Elements That Assist Initiation
Ex Biochem c24-promoter enhancer
24.15 Enhancers Contain Bidirectional Elements That Assist Initiation
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 indirectly with the promoter.

32. 24.16 Enhancers Contain the Same Elements That Are Found at Promoters
Ex Biochem c24-promoter enhancer
24.16 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

33. Ex Biochem c24-promoter enhancer
24.17 Enhancers Work by Increasing concentration of Activators Near 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 or
catenating the two molecules

34. Ex Biochem c24-promoter enhancer
Enhancer (DNA)  activator (蛋白質, 其他小分子, 代謝產物)
Silencer (DNA)  suppresor (蛋白質, 其他小分子, 代謝產物)