Promoters and Enhancers

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. 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. 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. 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. 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. Ex Biochem c24-promoter enhancer 24.4 RNA Polymerase I Has a Bipartite Promoter

13. 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. 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. Ex Biochem c24-promoter enhancer 24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters Figure 24.6

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

17. Ex Biochem c24-promoter enhancer

18. 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, +28 - +32 • A core promoter for RNA polymerase II includes: TATA box + Inr, or Inr + DPE

19. Ex Biochem c24-promoter enhancer Figure 24.10

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

24. 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. 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. 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. 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. 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 • https://www.youtube.com/watch?v=ysxtZJUeTCE • 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)