Chapter 20. Initiation of transcription. Eukaryotic transcription initiation

Transcription

1 Chapter 20. Initiation of transcription Eukaryotic transcription initiation

2 Prokaryotic vs eukaryotic Bacteria = one RNA polymerase Eukaryotes have three RNA polymerases (I, II, and III) in their nuclei. RNA polymerase II is used for mrna synthesis. Mitochondria and chloroplasts have their own RNA polymerases

3 Expression Control In Eukaryotes Some of the general methods used to control expression in prokaryotes are used in eukaryotes, but nothing resembling operons is known Eukaryotic genes are controlled individually and each gene has specific control sequences preceding the transcription start site In addition to controlling transcription, there are additional ways in which expression can be controlled in eukaryotes

4 Eukaryotes Have Large Complex Geneomes The human genome is about 3 x 10 9 base pairs or 1 m of DNA Because humans are diploid, each nucleus contains 6 x 10 9 base pairs or 2 m of DNA Some gene families are located close to one another on the same chromosome Genes with related functions appear to be distributed almost at random throughout the the genome

5 Highly Packaged DNA Cannot be Expressed Because of its size, eukaryotic DNA must be packaged Heterochromatin, the most highly packaged form of DNA, cannot be transcribed, therefore expression of genes is prevented Chromosome puffs on some insect chomosomes illustrate areas of active gene expression

6 Only a Subset of Genes is Expressed at any Given Time It takes lots of energy to express genes Thus it would be wasteful to express all genes all the time By differential expression of genes, cells can respond to changes in the environment Differential expression, allows cells to specialize in multicelled organisms. Differential expression also allows organisms to develop over time.

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8 General Factors Factors involved with transcription initiation Required for initiation at all promoters Join with RNA polymerase to form complex surrounding +1. General factors + RNAP = Basal transcription apparatus Upstream factors DNA binding proteins Recognize short consensus elements upstream of +1 Ubiquitous/not regulated Increase efficiency of promoter Inducible factors Function like upstream factors Have regulatory role regulate gene expression in time and space

9 Structure of eukaryotic promoter

10 Upstream elements Promoter elements Bind transcription factors No one element/factor absolutely required Protein-protein interactions are important. Upstream and inducible factors interact with basal transcription apparatus Promoter elements must be positioned near the +1

11 Enhancers Can be located several kb from promoter Can be present in either orientation relative to the promoter Contain elements that bind inducible factors Targets for tissue specific and/or temporal regulation

12 Transcription factors Any protein required for initiation but not a component of RNA polymerase is defined as a transcription factor. Many bind cis-acting elements in upstream region or enhancer. Some bind to other factors bound to upstream region or enhancer. Common mode of regulation is positive: activation of promoter.

13 Composition of eukaryotic RNA polymerases Large proteins - >500kD 8-14 subunits Three largest subunits similar to α, β, β from prokaryotic RNAP Three other subunits are common to all three RNAPs Largest subunit has carboxy-terminal domain (CTD) repeat of 7 amino acids (26-50 repeats)

14 Systems for defining promoters Oocyte system injection of DNA into nucleus Transfection systems exogenous DNA introduced into cultured cell Transgenic systems addition of gene to germ line In vitro reconstitution of initiation process with purified components

15 Analysis of promoters Deletion analysis progressive removal of sequences Determines boundary of promoter Point mutations mutations within boundary of promoters that lead to either increased or decreased rates of initiation Footprinting determines where transcription factor binds Determination of consensus sequences

16 How to identify promoter motifs Reporter gene eg CAT, luciferase Activity 100% Reporter gene 100% Reporter gene 20% Reporter gene 1% Reporter gene 0%

17 How to identify promoter motifs Reporter gene eg CAT, luciferase Activity 100% Reporter gene 100% Reporter gene 400% Reporter gene 1% Reporter gene 0%

18 RNA polymerase I has a bipartite promoter Core promoter: sufficient( ) Control element: efficiency control ( ) UBF1/ SL1 binds And then RNA polymerase I binding SL1: 4 proteins, TBP

19 RNA polymerase III uses both downstream and upstream promoters 5s and trna downstream snrna upstream TATA box TBP: preinitiation complex

20 The stages of reaction at internal promoters TFIIIB: true initiation factors positionaing factor - responsible for localizing RNA polymerase correctly TFIIIA, C: assembly factors

21 Eukaryotic transcription General Transcription factors Recognize and bind the promotor region, including at the TATA box Allow RNA polymerase to bind to promoter RNA polymerase + general transcription factors form transcription initiation complex. Transcription initiation complex Protein-protein interactions important between RNA polymerase and transcription factors

22 Basal transcription apparatus RNA polymerase + general factors General factors are involved in the process of binding RNAP to the DNA and initiating transcription. General factors TFIIX, where X is the letter (A, B, C, etc.) that identifies the factor

23 Generic promoter Initiator region Py 2 CAPy 5 (- 3-+5) TATA box ~ 25 bp upstream of +1 Only promoter element that is relatively fixed in relation to start point Tends to be surrounded by GCrich sequences Single base substitutions in TATA strong promoter down mutations Some promoters do not contain TATA

24 Step 1: binding of TFIID First step in initiation is binding of TFIID to TATA box TFIID TATA binding protein (TBP) + TAFs (TBP-associated factors) TBP is associated with different TAFs determines what class of promoters TBP will bind TBP binds minor groove of DNA and bends DNA ~ 80 o Only factor to make specific DNA contacts TAFs make extensive contact with DNA

25 3D structure of TBP & DNA Minor groove binding protein DNA bending

26 Step 2: binding of TFIIA TFIIA binds upstream of TFIID extension of footprint May act by relieving repression by TAF II 230 Step 3: binding of TFIIB TFIIB binds in vicinity of +1 (from 10 to +10) Likely interacts with RNA polymerase

27 Ternary complex of TFIIB-TBP-DNA

28 Step 4: Binding of RNAP-TFIIF TFIIF ATP dependent helicase (RAP74) RAP38 has homology to sigma factors TBP and TAFs may interact with CTD Initiation can take place at this time

29 Step 5: Binding of TFIIE TFIIE extends boundary upstream Required for promoter clearance Step 6: Binding of TFIIJ and TFIIH TFIIH ATPase, helicase, kinase of CTD

30 Phosphorylation of CTD required for promoter clearance

31 A connection between transcription and repair MFD TFIIH + kinase

32 Short sequence elements in upstream region TATA box(-35) CAAT box(-75) GC box(-90) Octamer

33 Mix and match A variety elements can contribute to promoter function, but none is essential for all promoters In individual promoter differ in number, location, and orientation

34 Common use of promoter elements Recognized by a corresponding transcription factor GC box- SP1 CAAT box- CTF, CDP, C/EBP Octamer- Oct1, Oct2