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General pattern of cis-acting control elements that regulate gene expression in yeast and metazoans
(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers as well as a TATA box or other promoter element. The latter positions RNA polymerase II to initiate transcription at the start site and influences the rate of transcription. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site.
Schematic diagram of the steps in the initiation of RNA synthesis (DNA transcription) catalyzed by RNA
polymerase.
The enzyme first forms a closed complex in which the two DNA strands remain fully base-paired. In the next step the enzyme catalyzes the opening of a little more than one turn of the DNA helix to form an open complex, in which the template DNA strand is exposed for the initiation of an RNA chain. The polymerase containing the bound subunit, however, behaves as though it is tethered to the promoter site: it seems unable to proceed with the elongation of the RNA chain and on its own frequently synthesizes and releases short RNA chains. As indicated, the conversion to an actively elongating polymerase requires the release of initiation factors (the sigma subunit in the case of the E. coli enzyme) and generally involves the binding of other proteins that serve as elongation factors.
Random transcription by bacterial RNA polymerase core
Promoter
E. coli RNA pol core
+
Random initiation
elongation
Transcripts of random size
'
'
'
'
'
'
Accurate initiation by bacterial RNA polymerase
Promoter
E. coli RNA polholoenzyme
+
initiation
elongationNascent RNA
Run-off transcript,Discrete size
+ +
'
'
RNA Pol core
Pol II basic promoter elements
- 10 0 -5 0 +1 5 0 100
core promote r
CAGAGC ATATAAGGTGAGGTAGG ATCAGTTGCTC CTCAC CTT -30 -20 -10 + 1
TA TA -box Inr
Random transcription by eukaryotic RNA polymerase
Promoter
Eukaryotic RNA pol II
+
Random initiation
elongation
Transcripts of random size
Fractionation of nuclear extracts to find GTFsFractionation scheme, DEAE cellulose
Matsui, Segall, Weil, Roeder (1980) JBC 255:11992
PlusPurifiedPol II
Run-offTranscriptAccurateInitiation at promoter
Accurate initiation by Euk RNA polymerase II plusfactors in the nucleus
Promoter +
initiation
elongation Nascent RNA
Run-off transcript,Discrete size
+ +
Eukaryotic RNA pol II + Nuclear extract, S-100
GeneralTranscription InitiationFactors
General transcription factors = GTFs• Proteins other than RNA polymerase involved in
transcription – Initiation, Elongation, Termination
• General transcription initiation factors (GTIFs)– Proteins required for specific transcription from
a minimal promoter (core) – Not subunits of purified RNA polymerase.– Required for RNA polymerase to bind avidly
and specifically to promoters.– GTIFs for RNA polymerase II are called TFIIx,
where x = A, B, D, …– Can have multiple subunits
GTFs for RNA polymerase II
TFIIDTBP }
TAFs
IIB
IIA IIE
IIF
IIH
helicase
protein kinase
TBP
InrIIB IIA Pol IIa
IIFIIE
IIH
CTD of large subunit of Pol II
Recognize core promoter
Targets Pol II to promoter
Modulates helicase
Helicase
CTD protein kinase
Many GTFs are possible targets for activators of transcription.
Sequential Binding
Model for assembly of preinitiation
complex
-30 +1
TATA Inr
Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA.
IIB
Eukaryotic RNA polymerase II
TFIID}TBP
TAFs
IIB
IIECTD of large subunit of Pol II
Pol IIa
or TBP
IIA
IIF
helicase
protein kinase
IIH
TATA Inr
IIA Pol IIa
IIFIIE
preinitiation complex
TATA Inr
IIAIIB Pol IIa
IIFIIE
ATP hydrolysis
initiation complex, DNA melted at Inr
IIH
IIH
= PIC
Activated PIC
TATA Binding Protein = TBP
• TBP binds in the narrow groove of DNA at the TATA box found about 20-25 bp 5’ to the start site for transcription of many (but not all) genes transcribed by RNA polymerase II.
• TBP bends the DNA about 90 degrees.• TBP alone or with TBP-associated proteins
(TAFs) plays an important role in recognizing the core promoter and recruiting RNA polymerase II to the promoter.
The Structure of 28kD TBP and TBP/DNA
TBP bends TBP bends DNA ~80DNA ~80oo and and forces open the forces open the minor groove.minor groove.
The association of TBP with TAFIs,TAFIIs, TAFIIIs, and PTF/SNAPc directs TBP to different promoter classes. The distribution of TBP among these factors contributes to the global regulation of gene expression. From Lee and Young (1998) Regulation of gene expression by TBP-associated proteins. Genes Dev. 12, 1398.
Four complexes function as class-specific promoter selectivity factors
TAFIIs of TFIID:-8 mostly conserved proteins ranging from 30 250 kDa
1. Strongly promote transcription from promoters with I (initiator) and D (downstream) elements.
2. X-linking (to DNA) and footprinting with different complexes showed that TAFII250 and TAFII150 bind Inr and D regions in cooperation with TBP.
Functions of TAFIIs (of TFIID):
TAFIIs also function to:
1. Promote transcription from some class II promoters that lack a TATA box.
2. Interact with some upstream activators (e.g., Sp1), and hence can act as co-activators.• Sp1 interacts with TAFII110• Gal4 NTF-1 activator works via TAFII150
and TAFII60
TFIIA and TFIIB
• TFIIA binds to TBP and could be considered a TAFII
• TFIIB is needed for the Pol/TFIIF complex to bind to TFIID, and can be though of as a linker between these two.
• A current model has TFIIA binding TBP on the upstream side, with TFIIB binding on the downstream side.
TFIIF
• 2 subunits, called RAP70 and RAP30 (for RNAP associated protein).
• Binds to the RNAP, and RAP30 delivers it to the DAB complex.
• Reduces non-specific binding of RNAP to DNA.
• Function is analogous to the factor in E. coli.
TFIIE and TFIIH• TFIIE
1. Binds after Pol/TFIIF binds to the pre- initiation complex.
2. Has 2 different subunits, both needed to stimulate transcription.
• TFIIH1. Required for promoter clearance 2. Complex protein with 9 subunits3. Has DNA helicase/ATPase activity (RAD25 gene)
for melting DNA at transcription bubble4. Also has Kinase activity: phosphorylates the
carboxyl terminal domain (CTD) of the large subunit of RNAP
TAFs are not universally required.
Based mostly on yeast strains with a temperature-sensitive TAFII subunit. RNA from each strain was hybridized to a microarray of 5500 yeast genes.
RNA Pol II bound to DNA and general transcription initiation factors
RNA polymerase II from yeast (does not include subunits 4 and 7)
136Ao
140AoA
o100 thick
Channel 25 Ao
diameter
Groove 25 Ao
wide
flexible end of "thumb"
Channel plus groove could accomodate 20-25 bp of duplex DNA
TFIIA
DNA
TBP
TFIIE
Darst, S.A., A.M. Edwards, E.W. Kubalek & R.D. Kornberg (1991) Cell 66: 121-128. Kornberg, R.D. (1996) Trends in Bioch. Sci. 21: 325-326.
16 resolutionAo
TFIIB
Model for RNA Polymerase II Phosphorylation
Eukaryotic RNA polymerase II
CTD of large subunit of Pol II
Pol IIa
CTD of large subunit of Pol IIP
PPP
PP
Pol IIokinase + ATP
phosphatase
Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation.
CTD has repeat of (YSPTSPT)26-50
RNAP making short RNAs, its stalled at +10 - +12.
TFIIH causes further DNA unwinding, allowing the bubble to grow and RNAP to go to elongation phase.
Other proteins involved in transcription and regulation
• In addition to RNA polymerase II and GTFs:• Proteins required for regulation, e.g.
– Gal11: regulation of the GAL operon– Rgr1: resistance to glucose repression
• Srb proteins– Yeast strains with truncations in the CTD of the large
subunit of RNA polymerase B are cold-sensitive– SRB genes: when mutated, suppress the phenotype of
CTD deletions– Extragenic suppressors: implicated in RNA polymerase
function
RNA polymerase II Holoenzyme and Mediator
• Holoenzyme– RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) +
(Rgr1, Gal11, others)– Correct initiation in presence of TBP (TFIID)– Responds to transcriptional activators
• Mediator– Complex needed for a response to transcriptional
activators by purified RNA Pol II plus GTFs– Yeast Mediator has 20 subunits, including Srb2, 4, 5, 6;
Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and others
• RNA Pol II + Mediator (+ some GTFs?) = Holoenzyme
Expanding the functions of RNA polymerase
Polymerase Trans-cribe
Start atPromoter
Respond toActivator
RNA Pol II Yes No NoRNA Pol II +GTFs
Yes Yes No
RNA Pol IIholoenzyme+ GTFs
Yes Yes Yes
The search for targets of transcription activators:the power of genetics + biochemistry
Roger Kornberg: purify “holoenzyme” from nuclear extractslook for a mediator of transcription factor activation in vitro
“Mediator” complex
Kim et al. (1994) Cell 77, 599.
"required reading"
EH
TBP
TAFB
activator
M
RNAPolII
F
M
M
elongation
A Model for assembly of Mediator-Holo RNA Polymerase II onto the DNA template
Stages in Initiation of Transcription• Bacterial
transcription• Closed complex:
holoenzyme+promoter• Open complex (DNA
melting, not need ATP)• Abortive transcripton• Productive initiation
– Transcribe past +9– Sigma dissociates
• Elongation
• Eukaryotic transcription
• Preinitiation complex (PIC) assembly
• PIC activation (DNA melting, needs ATP)
• Abortive transcription• Productive initiation
– CTD phosphorylated– Promoter clearance
• Elongation
Parallels between initiation pathway in prokaryotes and eukaryotes
From Eick et al. (1994)Trends in Genetics 10: 292-296
Enhancers and SilencersEnhancers and Silencers
• Enhancers stimulate transcription, silencers inhibit.
• Both are orientation independent.– Flip 180 degrees, no effect
• Both are position independent.– Can work at a distance from promoter– Enhancers have been found all over
• Bind regulated transcription factors.
An enhancer in an intron of a gamma-globulin gene.An enhancer in an intron of a gamma-globulin gene.
(a) Genes were constructed with the enhancer inverted (B), with it moved upstream of the gene (C) and inverted (D). The DNAs were transfected into mouse cells and synthesis of the protein was assessed by pulse-labeling with a radioactive amino acid, immunoprecipitation,
and separation by SDS-PAGE and autoradiography.
Four activators enriched in hepatocytes plus the ubiquitous AP1 factor bind to sites in the hepatocytespecific enhancer and promoter-proximal region of the TTR gene. The activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID, Srb/Mediator proteins, and general transcription factors, resulting in looping of the DNA and formation of a stable activated initiation complex. Because of the highly cooperative nature of complex assembly, an initiation complex does not form on the TTR promoter in intestinal epithelial cells, which contain only two of the four hepatocyte-enriched transcription factors. Many of the general transcription factors, Srb/Mediator proteins, and RNA polymerase II (Pol II) may be pre-assembled into a holoenzyme complex.
Model for cooperative assembly of an activated transcription-initiation complex at the TTR promoter in hepatocytes
Model of the enhancesome that forms on the -interferon enhancer.
Heterodimeric cJun/ATF-2, IRF-3, IRF-7, and NF-KB (a heterodimer of p50 and p65) bind to the four control elements in the ≈70-bp enhancer. Cooperative binding of these transcription factors is facilitated by HMGI, which binds to the minor groove of DNA. The cJun, ATF-2, p50, and p65 proteins all appear to interact directly with an HMGI bound adjacent to them. Bending of the enhancer sequence resulting from HMGI binding is critical to formation of an enhancesome. Different DNA-bending proteins act similarly at other enhancers. [Adapted from D. Thanos and T. Maniatis, 1995, Cell 83:1091 and M. A. Wathel et al., 1998,Mol. Cell 1:507.]
Sp1: Factor for Upstream (Proximal) Class II Promoter Element
• Binds GC boxes, stimulates transcription
• Interacts with TAFII110 in TFIID
• Also stimulates transcription of TATA-less nRNAP II promoter (by promoting TFIID binding)
Structure of Eukaryotic Transcription Factors
• Many have modular structure:1. DNA-binding domain
2. Transcription activating domain
• Proteins can have > 1 of each, and they can be in different positions
in protein.
• Many also have a dimerization domain
Regulation of galregulon in yeast
Regulation ofamino acidbiosynthesis inyeast
glucocorticoidreceptor: bindshormone and thenbinds DNA to altergene expression
general upstreamactivator of pol IIgenes binds the GCbox.
From Molecular Cell Biology 3rd edition, Lodish et al Scientific American Books 1995
N C
Gal4
GCN4
GR
SP1
N
N
N
C
C
C
DNA binding domainActivation domain
Recent data suggests SP1 actually has 4 activating domains.
Blau et al., Mol Cell Biol 1996, 16 (5): 204
Three functional Classes of Transcriptional Activation Domains
Required reading:
Activation Domains
1. Acidic (e.g., GAL4, 49 aa domain – 11 acidic aa)
2. Glutamine-rich (e.g., 2 in Sp1, ~25% gln)
3. Proline-rich (e.g., CTF, 84 aa domain – 19 are proline)
How do different trans-activation domains stimulates transcription?INITIATION VS ELONGATION
Three Functional classes of activation domains: