CLASS 3.5: 03/29/07 EUKARYOTIC TRANSCRIPTION I: PROMOTERS AND ENHANCERS

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1 CLASS 3.5: 03/29/07 EUKARYOTIC TRANSCRIPTION I: PROMOTERS AND ENHANCERS A. Promoters and Polymerases (RNA pols): 1. General characteristics - Initiation of transcription requires a. Transcription factors (TF) = - TFs may be proteins that: i. Recognize ii. Recognize iii. Recognize iv. Are incorporated b. Prokaryotic promoters - Recall that RNA pol binds - May need ancillary factors c. Eukaryotic promoters - Promoter is defined - These are recognized by - TFs required - RNA pol binds to startpoint, - RNA pol does not 1

2 d. Eukaryotic RNA polymerases - Divided into 3 classes i. RNA pol I = ii. RNA pol II = iii. RNA pol III = e. Enhancers - Promoter elements must - Enhancers = - Enhancers are often - Often are more densely - Proteins bound to enhancers - Difference between - Some sequence elements 2

3 2. Eukaryotic RNA Polymerases a. Have different locations in the cell i. Nucleolus = - site of ii. Nucleoplasm = - site for hnrna - site for iii. Mitochondria and chloroplasts - have - similar to b. Overall structure - Large - Each contains c. Specific subunits i. Some are homologous to ii. Some are iii. Carboxy-terminal domain (CTD) - found in - has Multiple repeats (26-50) of a consensus sequence = - CTD becomes 3

4 d. Sensitivity to poisons i. RNA pol II ii. RNA pol I iii. RNA pol III 3. Promoters for RNA pol I - Only 1 type of promoter - Makes most of the rrna - 5S rrna - RNA pol I exists as - Recruited as a. RNA Pol I promoter regions i. Core promoter - surrounds - most of region is - has conserved ii. Upstream promoter element (UPE) - extends from - also b. RNA Pol I ancillary factors i. Upstream Binding Factor (UBF) - needed for - binds - wraps DNA 360 so that 4

5 ii. SL1 - recruited to - responsible for ensuring that - has 4 subunits; 4. Promoters for RNA pol III a. Type of promoters i. Type 1 - has 2 short conserved sequences - TF III C and TF III A - TF III B (with positioning factor) - RNA pol - transcribes 5

6 ii. Type 2 - has 2 short conserved sequences - TF III C binds - TF III B (with positioning factor) - RNA pol - used to iii. Type III - found - look like - initiation can - initiation greatly increased by - used by 6

7 5. Promoters for RNA pol II - RNA pol II = - Basal factors (TF II X) = a. Core promoter - Shortest sequence in which - Minimum sequence needed - Functions only - Need additional factors - Components of the core promoter i. Startpoint - no - tendency for - called initiator sequence (InR) = ii. TATA box - located - core sequence = - TATA usually - TATA almost identical to - are some promoters 7

8 iii. DPE (downstream promoter element) - usually found - located - has - Core promoter either b. Core promoter binding proteins i. Positioning factor (TBP) - found in - TF II D has - TBP has saddle that - distorted TATA with TBP allows - in promoters without TATA, 8

9 ii. Basal apparatus binding factors - initiation requires - TF II A can - TF II B binds - TF II B determines - basal factor complex - RNA pol is now positioned, 9

10 iii. TF II H - TF II H has - TF II H associates - CTD tail must be a) clear promoter b) release some transcription factors c) recruit capping enzyme d) recruit SCAFs = e) recruit components of 10

11 - TFIIH has roles in - RNA pol stalls - RNA pol can use 11

12 iv. Activators - efficiency and specificity of how - activators are proteins - TATA sequence important for - activators influence the - types of activator sequences a) CAAT box - often - can be - can function b) GC box - often - consensus ~ - common to find - can be present - activator components of promoters have - no common element - fact that some can work in either direction suggests 12

13 B. Enhancers: 1. General characteristics - Promoters - Many cases activity of promoter - Characteristics distinguishing enhancers from promoters a. Position relative - promoters are - enhancer can be - can be b. Enhancers can - can be 2. Enhancer sequence elements - Tend to be - Many are - May differ from - Cooperative binding leads Example: binding of nonhistone protein (HMGI-Y) - Not like the mix and match promoters = - Proteins bound to enhancers - Create surface for 13

14 3. How enhancers work a. The distinction between i. Both have ability ii. Both are iii. Some are found iv. Some promoter elements b. Essential roles of enhancers - Increase the concentration of - Experiments have demonstrated that - Enhancer bound to proteins can - If close to 2 promoters, - Sometimes an insulator (DNA sequence that binds other proteins) can 14

15 4. CpG islands are regulatory targets a. General considerations - Recall that methylation of DNA - Methylation close to promoter - Demethylation would be required - CpG islands are - CpG islands are All housekeeping genes that are constitutively expressed Find CpG islands on b. Mechanisms by which CpG islands affect transcription i. Methylation for binding site of ii. Methylation may cause explains why active genes often 15