rna polymerases and general transcription factors final
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Transcription in Eukaryotes
Eukaryote RNA polymerases and their promoters
General transcription factors in Eukaryotes
Additional bibliography: Weaver, R. F. Molecular Biology, 2nd edition (2002)
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Evidences of multiple eukaryotic polymerases
Extracts of sea urchin embryos subjected to DEAE-sephadex chromatography
Incorporated
UMP
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Cellular localisation of the three RNA polymerases
Nucleoplasma fraction
Nucleolar fraction
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Subunit structure of yeast RNA polymerase II
Epitope taggingAn epitope is genetically added to the subunit Rpb3 of the yeast pol II
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Subunit structure of RNA polymerases from Saccharomyces cerevisiae
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The three eukaryote polymerases are related to the prokaryote polymerase
(and to one another)
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The three eukaryote RNA polymerases have different roles
Studiesin vitrowith purified RNA polymerases showed that they transcribespecific regions of the genomic DNA
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Effect of-amanitin on the activity of the three eukaryte RNA polymerases
Amanita phaloides
- is a poisonous mushroom
- produces -amanitin, a potent RNApolymerase II inhibitor
%maximala
ctivity
100
50
0
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Questions that you should be able to answer
at the end of this module:
1.How many polymerases are found in Eukaryotes?
2.What type of RNA is synthesized by each of these polymerases?
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Promoters: Class I, II and III
Enhancers
Silencers
How is RNA polymerase activity iniciated?
Different polymerases have different DNA binding sites (promoters)?
How is DNA transcription regulated?
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Class I promoters
RNA pol I transcribes only one gene - the rRNA percursor gene:
Example of the human rRNA gene:
2 critical regions sensitive to mutations(upstream control element and core element)
Spacing between these regions is very important
Exists in many copies in the genome
Is variable from species to species
Different species: differences in sequence, conserved architecture
-156 -107 -45 +1 +20
UCE Core
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Class III promoters
RNA pol III transcribes short genes, that encode small RNAs:5S rRNA, tRNAs, snRNAs
5S rRNA
tRNA orVA RNA
Intermediate element
Box A Box C
Box A Box B
Internal promoters: promoter sequences are found within the genes
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Exceptions to the internal promoter in genes transcribed by RNA pol III
7SL gene
Codes for the major component of the Signal Recognition Particle (SRP)
Has a weak internal promoter and a 5flanking region is required for high-level transcription
U6 RNA gene
Has no internal promoters; the 5flancking region resemblesClass II promoters (contains a TATA box)
Homework:What is the SRP?
Homework:Function of U6 RNA?
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Most common element in the promoteris the TATAAA consensus sequence
Class II promoters
Present in genes that code for proteins and snRNA genes
TATA box
Other TATA box sequences may be found due to sequence variation
T frequently replace A
G and C can substitute nucleotides in the TATA box
example: the rabbit-globin gene promoter starts with CATA
GATA is another variation found in class II promoters
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Variable location;
In yeast, it can be found from50 to 70 bp upstream thetransciption start site
Class II promoters: core promoter + upstream element
TATA box (positioned around -25 nt )Core promoter Initiator, centered on the transcription start site
Downstream element
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What is the function of the TATA box?
Effects of deletions in the histone H2A promoter of the sea urchin
Specialised genes have TATA boxes
Genes that are expressed only in certain types of cells (ex: keratin in skin
cells; hemoglobin in red blood cells) or in certain environmental conditions
Removing the TATA boxcauses transcription to initiateat a wide variety of sites
A B C
TATA
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The TATA box is also involved in positioning the transcription start site
Effect of deletions in the SV40 (simian virus 40) promoter:
The distance between the TATA box and the transcription initiation siteremained constant
Transcription started about 30 bp downstream of the first T of the TATA box (usually with a purine)
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In some promoters, removal of the TATA box impairs promoter function
Removal of the TATA box of the rabbit-globin gene inhibits transcription
Wtfragment
Shorterfragment
1
2
3
wt
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Molecular Biology technique 1: S1 Nuclease Protection Assay
Probe molecules or RNA moleculesthat do not hybridize are removed byS1 nuclease digestion
The hybridmolecules areethanol-precipitated
Recovered hybridmolecules areseparated on adenaturingpolyacrylamide-gel followed byautoradiography
The size and abundance of the protected moleculesis a direct measure of the steady-state level for a specific RNA
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Molecular Biology technique 2: Primer extension
Transcription
Hybridize labeled primer
Extend primer with reverse transcription
DNA
RNA
Denature hybridElectrophoresis
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Genes transcribed by RNA Pol II may not have TATA box promoters
Constitutively active. Found in housekeeping genesTranscription is regulated by Sp1 (Specific protein 1)Ex: adenine deaminase, part of the nucleotide synthesis pathway
TATA-less promoters
- GC boxes: GGGCCC or CCGCCC
- CCAAT box
- Other initiator sequences
Homeotic genes (regulated during development)
Ex:Genes that control the development of the fruit fly, or genes activated during the developmentof the immune system in mammals. Ex:TdT gene (mouse terminal deoxynucleotidyl transferase)contains a 17 bp initiator sequence sufficient to drive basal transcription
Transcription is regulated byCCAAT-binding transcription factor (CTF)and CCAAT-enhancer binding protein (C/EBP)Ex: Herpes simplex virus (HSV) timidine kinase promoter
HSV
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Some need the TATA box for proper functioningEx: rabbit -globin gene promoter
Some need the TATA box for correct positioning of the Pol II
at the transcription start siteEx: SV40 (simian virus 40) promoter
Some dont have a TATA box and are functional
(TATA-less promoters)Ex: GC boxes and CCAAT box
Important concepts regarding Class II promoters
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Transcription initiation depends on the formation of a protein complex
The pre-initiation complex
In Class II promoters
The preinitiation complex is assembled at the TATA box position
Assembly is mediated by TBP, which recognises the TATA boxand allows the condensation of other protein factors
TBP-associated factors, TAFs or TAFIIs
A large multi-component complex in
which RNA polimerase interactswith accessory proteins known asthe general transcription factors(GTFs)
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Molecular model of the TATA box-binding protein (blue and purple)complexed with DNA (pink).
The TATA box is recongnised by TBP - TATA box Binding Protein
- Is highly conserved in eukaryotes
-Binds to the minor groove
of the TATA box
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Structure and composition of TFIID
Hypothetical structure of thereconstituted DrosophilaTFIID
Gel electrophoresis of in vitroreconstituted TFIID from Drosophila.
Eight major TAFIIs are visible in thegel.
TFIID composition is highlyconserved from yeast to man
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How is the preinitiation complex formed in TATA-less promoters?
TBP association with the TATA box
Complex with all the TAFII factorsthat form TFIID
Complex with a sub-set of TAFII factors
TAFII250 and TAFII150 are sufficient to
bring TBP to the Initiator sequence
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TFIID association with GC box promoters
in the complete TFIID complex
The complex formed byTFII250, TFII150 and TFII150is sufficient to anchor TBP to Sp1.
TAFII110 interacts with Sp1
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Different TAFII combinations respond to different activators
TAFII250 and TAFII110 promote the
interaction between TFIID and Sp1,the protein that binds to GC boxes
TAFII250 and TAFII150 mediate
the association of TFIID to the
Initiator sequence and to
downsteam elementsTAFII250 is an histone acetyl-
transferase and a kinase;
modifies histones and other GTF
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A model for transcripton enhancement by activators
TFIID supports activation by several activators
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TATA
Start
Downstreamelement
GCbox
CCAATbox
Recognisedby Sp1
Recognised byCTFC/EBP
The rate of transcription is regulated by promoter proximal elements
Trans-acting elementsactivator proteins (DNA binding domain + activator domain)
Stabilize the preinitiation complex, increasing the frequency of transcription
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GC boxes may also work as transcription stimulators
- Orientation-independent, like enhancersthey can be flipped 180 and still function
- Position-dependent, like other promotersif they are moved more than a few base pairs away from the TATA box they loose theability to stimulate transcription
A specific transcription factor, known as
Specificity Protein 1 (Sp1)
binds to the GC boxes and stimulates transcription
GC boxes are
Sp1
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Tissue specific effectThey depend on the activity of DNA binding proteins that are specific to a given tissue
Enhancers - stimulate
Silencers - repress
Silencers may partially or completely repress transcription
High levels of transcription; enhancers work in either orientation
They are position and orientation independent
unlike promoters, which are position and orientation dependent!
Cis-acting elements: distal DNA elements that strongly influence transcription
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TFIID functions
Recognition of DNA sequence elements of the core promotermay be mediated directly by some TAFIIs or may be activator-dependent(Sp1 mediates the link to GC boxes)
Generation of a chromatin environment favourable for transcription initiation
Induces structural modifications in other general transcription factors, facilitating the assembly
of the pre-initiation complex and transcription initiation
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Several GTPs compose the pre-initiation complex
Factor Number ofsubunits
MW(kD)
Function
TBP
TFIID-TAFs
TFIIA
TFIIB
TFIIF
TFIIE
TFIIH
1
12
3
1
2
2
9
38
15-250
12, 19, 35
35
30, 74
34, 57
89, 80, 62,52, 44, 34,32, 38, 40
Recognize core promoter
Recruit TFIIB
Assist transcription activationAssist promoter recognition
Stabilize TFIID and promoter binding
Recruit RNA Pol II and TFIIF
Assist RNA Pol II to reach promoter
Promoter meltingRecruit TFIIH and modulate its helicase,ATPase and kinase activities
Phosphorilation of the CTD of RNA pol IIPromoter melting using helicase activity
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Formation of the DABPolFEH pre-initiation complex(from in vitroexperiments)
TFIID + TFIIA + TFIIB
DAB complex is assembled on the TATA box(TFIIA is optional in vitro)
1)
2) TFIIF binds to polymerase and leads it to the DAB complex
DABPolF complex
3)
E
H
DABPolFEH complex
Finally, TFIIE and TFIIH join the DABPolF complex
DABPolF + TFIIE + TFIIH
DAB complex
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General transcription factor requirements in Yeast
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Factors involved in the regulation of transcription by RNA Pol II
Long regions of the DNA can loop over to enable the regulatory connections
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Questions that you should be able to answer
at the end of this module:
1. Which are the main elements of Class I, II e III promoters? Where are they localized?
2. What is the function of the TATA box in class II promoters? Which protein recognises the TATA box?
3. How is a pre-initiation complex formed in class II promoters that have TATA box?And in promoters that dont have a TATA box?
4. What is the function of the GC box in class II promoters? Which protein recognises the GC box?
5. Beyond TATA boxes and GC boxes, what other motifs can be found in class II promoters?
6. What is the function of the Initiator in class II promoters?
7. What are proximal activator sequences? Where are they located and how do they work?
8. What are enhancer and silencer sequences? Where are they located and how do they work?
9. What is the DABPolFEH complex? What is the function of the DABPolFEH complex?
10. What is the function of the general transcription factors TFIIB, TFIID, TFIIF, TFIIE and TFIIH?
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Stabilises TFIID
in the promoter
Links TFIID and TFIIF/Pol II,promoting the assembly of thecomplete pre-initiation complex
Directs RNA Pol II to promoterswith assembled DAB complex
Reduces non-specific interactions
between RNA Pol II and the DNA
TFIIH
TFIIE
recruits TFIIHzinc finger domain binds to DNA
TFIIF
Phosphorylation of RNA Pol II
Promoter melting throughhelicase activity
TFIIA
TFIIB
A model for the pre-initiation complex
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TFIIS stimulates RNA Pol II proofreading of transcripts
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TFIIS stimulates RNA Pol II proofreading of transcripts
Incorrect nucleotides (in yellow)may be incorporated by RNA Pol II
RNA Pol II backtracks, extrudingthe 3 end from the active site
The RNAse activity of the RNA Pol IIeliminates the incorrect nucleotide
RNA Pol II resumes activity
Th RNA P l II h l
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Core
Holoenzyme
The RNA Pol II holoenzyme
The RNA Pol II holoenzyme is a complex formed byRNA Pol II and a subset of initiation factors, found notlinked to the promoter
In yeast, the RNA Pol II holoenzyme contains a subset of andregulatory proteins (SRB/mediator proteins) and generaltranscription factors, including TFIIF
The composition of the holoenzyme depends onthe type and physiological state of the cell
In mouse liver cells, the RNA Pol II holoenzyme containsTFIID, B, E and F
Preinitiation complex formation at Class I promoters
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Preinitiation complex formation at Class I promoters
SL1 interacts with the RNA Pol Ito strengthen the binding of thecomplex to the UCE
UBF stimulates transcriptionthrough interactionwith the UCE
TFIA is essentialfor RNA Pol Irecruitment
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TFIIIA is a zinc-finger protein
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http://www.youtube.com/watch?v=GRL_rdB30GY&feature=related
Homework:
video on YouTube
It is composed of 9 zinc-fingers in tandem repeatinteracts with the DNA through the major groove
Zinc-finger structural motif-helix
-sheetZinc
The order of binding of transcription factors is important
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5S rRNA
The order of binding of transcription factors is important
5S rRNA gene transcription only occurres whenTFIIIB is added after TFIIIA and TFIIIC
TFIIIB cannot bind to the promoter by itself;only binds if TFIIIC and TFIIIA are already present
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Molecular Biology technique 3: DNA footprinting
DNA containing a transcription factor binding site
Transcription factor
Radioactive label
footprint
fragments
Electrophoresisand autoradiogram
Finding the DNA sequence targeted by a DNA binding protein
DNAse I digestion under mild conditions(approximately 1 cut per DNA molecule)
TFIIIB and TFIIIC DNA binding sites in a tRNA gene
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TFIIIB and TFIIIC DNA binding sites in a tRNA gene
DNA footprinting using a labeled tRNA gene andcombinations of purified TFIIIB and TFIIIC
In d, when the sample was treated with heparin to
strip off loosely bound protein, TFIIIB remainsbound to the upstream region but TFIIIC wasremoved
In b, TFIIIC protects the internal promoter, especiallyBox B, but not the upstream region
In c, when TFIIIC and TFIIIB are present, theupstream region is also protected
TFIIIC
TFIIIB
TFIIIC
TBP plays a role in the formation of the preinitiation
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complex in Class III promoters
TFIIIC organizes the formation of thepreinitiation complex, promoting thebinding of TFIIIB (with TBP) in theupstream region
TFIIIB promotes the binding of RNAPol III at the Start site
As RNA Pol III proceeds, TFIIIC isremoved but TFIIIB remains in place
TFIIIB is composed of TBP andtwo TAFs: TAF-172 and TAF-L
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Questions that you should be able to answer
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yat the end of this module:
1. O que o complexo DAB? Qual a sua funo?
2. Como constitudo o complexo DABPolFEH?
3. Qual o significado funcional da actividade de acetil-transferase de histonas e de cinase de TAFII250?
4. Nos promotores sem TATA como que se liga o complexo TFIID ao promotor?
5. Qual a funo das caixas GC na ligao do complexo TFIID ao promotor?
6. Qual a funo de TFIIA e de TFIIB na formao do complexo de pr-iniciao? e qual a funo dosfactores TFIIF, TFIIE e de TFIIH?
7. Qual o significado funcional da fosforilao da terminal carboxlico da RNA polimerase II (CTD)?
8. Qual a funo dos factores de alongamento da transcrio?
9. Quais os factores gerais da transcrio dos genes da Classe-I?
10. Qual a funo e como constitudo o complexo SL1?
11. Qual a funo do complexo UBF? Com o funciona?
12. Quais so os factores gerais da transcrio dos genes da Classe-III?
13. Como funcionam os factores TFIIIA, TFIIIB e TFIIIC?
14. Qual a funo da TBP na formao do complexo de pr-iniciao das classes I, II e III?