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Promoters and Enhancers. Chapter 24. 24.1 Introduction. Figure 24.1. 24.2 Eukaryotic RNA Polymerases Consist of Many Subunits. RNA polymerase I synthesizes rRNA in the nucleolus. RNA polymerase II synthesizes mRNA in the nucleoplasm.
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Promoters and Enhancers Chapter 24
24.1 Introduction Figure 24.1
24.2 Eukaryotic RNA Polymerases Consist of Many Subunits • RNA polymerase I synthesizes rRNA in the nucleolus. • RNA polymerase II synthesizes mRNA in the nucleoplasm. • RNA polymerase III synthesizes small RNAs in the nucleoplasm.
All eukaryotic RNA polymerases have ∼12 subunits and are aggregates of >500 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. Figure 24.2
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
24.4 RNA Polymerase I Has a Bipartite Promoter • The RNA polymerase I promoter consists of: • a core promoter • an upstream control element (UPE) Figure 24.5
The factor UBF1 wraps DNA around a protein structure to bring the core and UPE 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.
24.5 RNA Polymerase III Uses Both Downstream and Upstream Promoters • RNA polymerase III has two types of promoters. Figure 24.7
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
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. • TFIIIB has TBP as one subunit and enables RNA polymerase to bind. Figure 24.9
24.7 The Startpoint for RNA Polymerase II • RNA polymerase II requires general transcription factors (called TFIIX) to initiate transcription. • RNA polymerase II promoters have a short conserved sequence Py2CAPy5 (the initiator InR) at the 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 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 • either a TATA box or a DPE Figure 24.10
24.8 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 TAFs • The total mass is ∼800 kD. Figure 24.11
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.
24.10 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
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 elongationto begin. • Further phosphorylation of the CTD is required at some promoters to end abortive initiation. Figure 24.17
24.12 A Connection between Transcription and Repair • Transcribed genes are preferentially repaired when DNA damage occurs. Figure 24.19
TFIIH provides the link to a complex of repair enzymes. • Mutations in the XPD component of TFIIH cause three types of human diseases Figure 24.20
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. • The factors that bind to them to stimulate transcription are called activators.
24.14 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
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
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.
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
24.17 Enhancers Work by Increasing theConcentration 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 or • catenating the two molecules
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
24.18 Gene Expression Is Associated with Demethylation • Demethylation at the 5′ end of the gene is necessary for transcription.
24.19 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.
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.