Preinitiation complex

In eukaryotic transcription describes the assembly of transcription factors at the promoter before RNA polymerase binds

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The startpoint for RNA polymerase II

eRNA polymerase II requires general transcription factors (TFIIX) to initiate transcription Basal transcription apparatus: RNA polymerase II + TFIIXs Subunits of RNA polymerase II and general transcription factors are conserved among eukaryotes
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Core promoter

Shortest sequence at which RNA polymerase II can initiate transcription Can in principle be expressed in any cell Minimum sequence that enables the general transcription factors to assemble at the startpoint
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Core promoter

Core promoter functions at only a low efficiency. Activators are required for a proper level of function

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Homologies in the regions near the startpoint are restricted to short sequences.

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Minimal pol II promoter

Has a TATA box ~25 bp upstream of the InR TATA box =consensus sequence= TATAA

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TATA BOX

TATAA usually followed by three more AT base pairs Surrounded by GC-rich sequences Almost identical with the 10 sequence found in bacterial promoters It could pass for bacterial one except for the difference in localitation at 25 instead of 10

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TATA BOX

Single base substitutions in the TATA box act as strong down mutations. Some mutations reverse the orientation of an AT pair, so base composition alone is not sufficient for its function. So the TATA box comprises an element whose behavior is analogous to our concept of the bacterial 4/24/12 promoter: a short, well-defined

TATA-less promoters

Promoters that do not contain a TATA element 50% or more of promoters may be TATA-less When a promoter does not contain a TATA box, it usually contains another element: DPE (downstream promoter element) which is located at +28 to 4/24/12 +32

Minimal pol II promoter

Has a initiator (Inr) = Py2CAPy5 positions 3 and +5 Inr = pyrimidines (Y) surrounding startpoint Tendency for first base of mRNA to be A flanked on either side by pyrimidines

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Core promoter

Can consist: TATA box + InR InR + DPE

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To recognice a promoter containing a TATA box

The first step is: Binding of factor TFIID (~800 kD) TFIID contains:

TATA-binding protein (TBP)

~30-250 kD Subunit of TFIID Binds to DNA

11 TAFs: (TBP-associated factors)

tissue-specific.

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Binding of TFIID to the TATA box is the first step in initiation. Other transcription factors bind to the complex in a defined order, extending the length of the protected region on DNA. When RNA polymerase II binds to the complex, it initiates transcription.
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The basal apparatus assembles at the promoter

TBP is a universal factor

TBP is the key component Incorporated at each type of promoter by a different mechanism Positioning factors that consist of TBP associated with a set of TAFs are responsible for identifying all classes of promoters RNA polymerase II: positioned in a fixed distance of the TATA box from 4/24/12 startpoint

RNA pol is positioned at promoters by a factor that contains TBP.

Promoters for RNA polymerase III: TFIIIB binds adjacent to TFIIIC. Promoters for RNA polymerase I: SL1 binds in conjunction with UBF. Promoters for RNA 4/24/12 polymerase II: TFIID

Positioning factor recognizes promoter in a different way

In TATA-promoters TBP binds specifically to DNA In other promoters TBP is associated with other proteins that bind to DNA It has the common purpose of interaction with the RNA polymerase

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TFIID is ubiquitous, but not unique.

All multicellular eukaryotes also express an alternative complex, which has TLF (TBP like factor) instead of TBP TLF is ~60% similar to TBP TLF no reconocen cajas TATA

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TBP has the unusual property of binding to DNA in the minor groove. (Virtually all known DNA-binding proteins bind in the major groove.)

TBP binds DNA in an unusual way

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TBP binds to the minor groove

TBP surrounds one face of DNA, forming a "saddle" around the double helix DNA-binding site consists of a C-terminal domain N-terminal tail is variable and interact with other proteins

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TBP surrounds DNA

TBP surrounds DNA from the side of the narrow groove TBP consists of two related (40% identical) conserved domains (light and dark blue) The N-terminal region varies (green) The two strands of the DNA double helix are in light and dark grey.
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Nucleosomes prevents transcription

Nucleosomes form preferentially by placing AT-rich sequences with the minor grooves facing inward They prevent binding of TBP

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Structure implications

Allows the transcription factors and RNA polymerase to form a closer association than would be possible on linear DNA. TBP in the minor groove + other proteins in the major groove = high density of protein-DNA contacts in this region
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Binding of purified TBP to DNA in vitro protects ~1 turn of the double helix at the TATA box, typically extending from 37 to 25 Binding of the TFIID complex in the initiation reaction regularly protects the region from 45 to 10
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TBP is the only general transcription factor that makes sequence-specific contacts with DNA

TFIID

Within TFIID as a free protein complex; Factor TAFII230 binds to TBP TAFs : TAFII42 and TAFII62 Together with other TAFs, TAF II42 and TAFII62 form the basis for a structure responsible for the nonsequence-specific interactions of TFIID with DNA.
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Initiation requires

Transcription factors to act in a defined order : The order was determined by Footprinting of DNA protected by each complex As each TFII factor joins the complex, an increasing length of DNA is covered. RNA polymerase is incorporated at a late stage
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Adition order

Complex: TFIID binds the TATA box. (TFIID also recognizes the
InR sequence at the startpoint.)

TFIIA joins the complex

(TFIIA may activate TBP by relieving the repression that is caused by the TAFII230)

TFIIB is bound downstream of the TATA box 4/24/12

TFIIB

TFIIB contacts the minor groove downstream of TATA box TFIIB contacts the major groove upstream of the TATA box (region BRE) TFIIB is responsible for the directionality of RNA polymerase
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Complejo Ternario

Ternary complex: TFIIB-TBP-DNA TFIIB binds along the bent face of DNA The two strands of DNA are green and yellow TBP is blue TFIIB is red and purple 4/24/12

TFIIB interacts with RNA polymerase

N-terminal zinc ribbon from TFIIB contacts the enzyme near the site where RNA exits An elongated "finger" of TFIIB is inserted into the polymerase active center. The C-terminal 4/24/12

TFIIB binds to DNA and contacts RNA Pol

N-terminal zinc ribbon from TFIIB contacts the enzyme near the site where RNA exits An elongated "finger" of TFIIB is inserted into the polymerase active center. The C-terminal domain 4/24/12 interacts with the RNA

TFIIF

TFIIF : heterotetramer consisting of two types of subunit The larger subunit : RAP74 ATP-dependent DNA helicase activity Involved in melting the DNA at initiation. The smaller subunit: RAP38
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Binds tightly to RNA polymerase II

Binding of the RNA Pol II

Complex of TBP and TAFs interact with the CTD tail of RNA polymerase Polymerase binding downstream from +15 on the template strand and +20 on the nontemplate strand

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What happens at TATA-less promoters?

The same general transcription factors Inr provides the positioning element TFIID binds to it via an ability of one or more of the TAFs to recognize the Inr directly TAFs in TFIID also recognize DPE element downstream the startpoint
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Contrast with prokaryotic transcription:

Bacterial RNA polymerase has an intrinsic ability to bind DNA Sigma factor becomes part of the RNA Pol II before DNA is bound

Needed for initiation but not for elongation Later released

RNA polymerase II can bind to the promoter only after separate 4/24/12 transcription factors have bound

Eucariotic vs. Procariotic

Basal apparatus assembles at promoter sequences Factors are primarily responsible for the specificity of promoter recognition Some of the factors participate in protein-DNA contacts (only TBP makes
sequence-specific contacts)

Protein-protein interactions are 4/24/12 important in the assembly of the

TATA deletion

Causes the site of initiation to become erratic Reduces relatively few transcription

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TATA box (if present) determines the location of the startpoint

TATA-less promoters lack unique startpoints Initiation occurs at any one of a cluster of startpoints TATA box aligns the RNA polymerase (via TFIID and other factors) to initiates at the proper site
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This explains fixed location of TATA

Activators

Assembly take place at the core promoter in vitro, but is not sufficient for transcription in vivo Interactions with activators are required Activators recognize upstream elements
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Activators interact with the basal

Initiation is followed by promoter clearance

TFIIE and TFIIH are required to melt DNA to allow polymerase movement CTD Phosphorylation is required for elongation RNA Pol II CTD may coordinate processing of RNA with transcription
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Some Transcription factors act at a later stage

Binding of TFIIE causes the boundary of the region protected downstream to be extended by another turn of the double helix, to +30 TFIIH and TFIIJ join the complex after TFIIE They do not change the pattern of binding to DNA
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TFIIH is an exceptional factor that may play a role in elongation

TFIIH is the only general transcription factor that has independent enzymatic activities It include: ATPase Helicases of both polarities

Kinase activity that can phosphorylate the CTD tail of RNA polymerase II 4/24/12

The initiation reaction

Defined by formation of the first phosphodiester bond Occurs once RNA polymerase has bound Phosphorylation of the tail is needed to release RNA polymerase II from the transcription factors Most of the transcription factors are released from the promoter at this 4/24/12 stage

Phosphorylation of the CTD by the kinase activity of TFIIH is needed to release RNA polymerase to start transcription.

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TFIIE and TFIIH are required to melt DNA

On a linear template, ATP hydrolysis, TFIIE, and the helicase activity of TFIIH (provided by the XPB subunit) are required for polymerase movement Helicase activity of the XPB subunit of TFIIH is responsible for the actual melting of DNA
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RNA polymerase II stutters at some genes when it starts transcription

Short RNA product is degraded rapidly A kinase called P-TEFb is required to extend elongation into the gene This is a member of the cdk family
(cell cycle)
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RNA polymerase II terminates after a short distance

CTD is required for:

The capping

guanylyl transferase binds to phosphorylated CTD: important to modify the 5 end as soon as it is synthesized.

Splicing:
Proteins called SCAFs bind to the CTD, and they bind to splicing factors; coordination of transcription and 4/24/12

Initiation in bacterias and eucariotes

In bacteria: formation of the open complex completes the necessary structural change to DNA In eukaryotics: unwinding of the template is needed after this stage

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A connection between transcription and repair

TFIIH provides the link to a complex of repair enzymes Mutations in the XPD component of TFIIH cause three types of human diseases

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A connection between transcription and repair

Direct link from RNA polymerase to repair activation Transcribed genes are preferentially repaired when DNA damage occurs Only template strand of DNA is rapidly repair Nontemplate strand is repaired at the same rate as bulk DNA
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Repair

In bacteria: uvr excision-repair system Preferential repair is abolished by mutations in the gene mfd This provides the link from RNA polymerase 4/24/12 to Uvr enzymes

Roles of Mfd protein

1.

Displaces ternary complex of RNA polymerase from DNA Causes the UvrABC enzyme to bind to the damaged DNA This leads to repair of DNA by the excisionrepair mechanism

2.

3.

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Corrections in eucariotes

Template strand of a transcribed gene is preferentially repaired General transcription factor TFIIH is involved (transcription and repairing damage) Helicase subunit (XPD) creates the initial transcription bubble and melts

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TFIIH

Common function: Initiating transcription and repairing damage The same helicase subunit (XPD) creates the initial transcription bubble and melts DNA at a damaged site
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TFIIH

Associated with other subunits that have a kinase activity; this complex also includes a repair subunit. Kinase catalytic subunit that phosphorylates the CTD of RNA polymerase belongs to a group of kinases

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The TFIIH core may associate with a kinase at initiation and associate with a repair complex when damaged

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The alternative complex

Consists of the core associated with a large group of proteins that are coded by repair genes. Other proteins associated with the complex include endonucleases (XPG, XPF, ERCC1) Subunits with the name XP are coded by genes in which mutations cause the disease xeroderma pigmentosum
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TFIIH dissociates from RNA polymerase at an early stage of elongation (after transcription of ~50 bp) The large subunit of RNA polymerase is degraded when the enzyme stalls at sites of UV damage.

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Cockayne's syndrome

Is caused by mutations in either of two genes (CSA and CSB), both of whose products appear to be part of or bound to TFIIH. Occasionally caused by mutations in XPD it involves mental retardation and is marked by changes in the structure of hair

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Trichothiodystrophy

Caused by mutations in XPD Which has little in common with XP or Cockayne's Mutations that prevent XPD from stabilizing the complex cause trichothiodystrophy. The helicase activity is required for the 4/24/12 repair function.

Short sequence elements bind activators

Activator: Protein that stimulates the expression of a gene Act at a promoter to stimulate RNA polymerase In eukaryotes, the sequence to which it binds in the promoter is called a response element. 4/24/12

Short sequence elements bind activators

CAAT box is part of a conserved sequence located upstream of the startpoints of eukaryotic transcription units; it is recognized by a large group of transcription factors. The GC box is a common pol II promoter element consisting of the sequence GGGCGG. Short upstream elements increase 4/24/12 frequency of initiation. the

Startpoint:

A promoter for RNA polymerase II consists of two types of region

itself is identified by the Inr and/or by the TATA box close by. In conjunction with the general transcription factors, RNA polymerase II forms an initiation complex surrounding the startpoint, as we have just described. The efficiency 4/24/12 and specificity

An analysis of a typical promoter is summarized in Figure 21.21. Individual base substitutions were introduced at almost every position in the 100 bp upstream of the -globin startpoint. The striking result is that most mutations do not affect the ability
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of the promoter to initiate

Saturation mutagenesis of the upstream region of the -globin promoter identifies three short regions (centered at -30, -75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, and GC boxes.

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The TATA box (centered at 30) is the least effective component of the promoter as measured by the reduction in transcription that is caused by mutations. But although initiation is not prevented when a TATA box is mutated, the startpoint varies from its
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usual precise location. This confirms

The sequence at 75 is the CAAT box. Named for its consensus sequence, it was one of the first common elements to be described. It is often located close to 80, but it can function at distances that vary considerably from the startpoint. It functions in
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either orientation. Susceptibility to