general pattern of cis-acting control elements that regulate gene expression in yeast and metazoans...

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: