Saturday, 22 December 2012

RNAPII Recruitment and Transcript initiation

Interaction of TFIIB zinc ribbon with RNAPII by Elsby and Roberts
TFIIB contains a zinc ribbon at N-terminus. Between zinc ribbon and core domain lies most conserved TFIIB region. It is termed the B-finger and a linker region.  B finger is within pol II catalytic centre. When assembled to promoter, it is close to the region of transcription initiation. This spports observations that mutations in B finger can cause change in TSS.

Zinc ribbon is required to recruit Pol II to promoter.  TFIIB zinc finger mutants C34A, G30A, D31A and V40A fail to recruit TFIIB.

Zinc ribbon domain contacts dock domain of Pol II subunit Rpb1 between residues 409 and 419.

Mutations in TFIIB B finger often shift TSS or block transcription that occurs after recruitment of PolII to promoter. TFIIB B finger projects into catalytic centre of Pol II, suggesting that B finger substitution might affect activity. B finger substitutions have been found to affect promoter clearance.

RNA polymerase II-TFIIB structure and mechanism of transcription initiation by Kostrewa et al
RNAPII-B complex was crystallised.

B-reader and DNA start site scanning
After open complex formation, yeast Pol II scans DNA for Inr that defines TSS.  Flexible B-reader loop may help recognise Inr nt and stabilise first 2 NTP substrates in active site in initiating complex. Arg 78 mutation causes TSS shirts and altered abortive transcription in vitro.

Initiation-elongation transition
Promoter DNA is recruited to Pol II by B-ribbon that binds dock. Promoter DNA is positioned over the Pol II active centre cleft with B-core domain that binds wall at end of cleft.

DNA is melted 20bp downstream ot TATA box with help of B linker.  Emerging template strand slips in cleft to fill template tunnel. Bubble is stabilised near active centre with help of B reader. Downstream DNA duplex is loaded into downstream cleft. (Open complex formation).

Template strand is threaded through template tunnel along active site. It is scanned for Inr motif with B-reader (DNA start site scanning).

First 2 NTPs are positioned opposite a conserved Inr dinucleotide motif and first phosphodiester bond is formed. (RNA chain initiation.)

RNA growth causes short-DNA-RNA hybrids that are transiently bound. Causing frequent ejection of short RNAs (abortive transcription).

Growth of RNA beyond 7nt triggers B release and formation of elongation complex (promoter escape).

B release results from a clash of RNA with B-reader helix, and from a clash of upstream DNA duplex with non-template DNA in elongation complex with B-linker above rudder. B release during promoter escape requires an RNA length of at least 7 nt and rewinding of upstream DNA.

Linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions by Wiesler
A few deletions cause superstimulation. Abortive transcription is stimulated at a high level. 

Single amino acid residues influence recruitment of TFIIB-RNAP complex.  Abortive transcription rate can be regulated by length of a polypeptide stretch in B linker region. 

The Role of the transcription bubble and TFIIB in promoter clearance by RNAPII by Pal et al
Regardless of promoter spacing, upstream edge of bubble forms 20bp from TATA. It expands downstream until 18 bases are unwound and RNA is at least 7 nt long. At this point upstream 8 bases if bubble reanneal (bubble collapse). If bubble size or transcript length is insufficient, bubble collapse cannot occur.  Stability decreases with increasing bubble size up to bubble collapse. After that stability is restored.  

Bubble collapse suppresses pausing at +7 to +9 associated with presence of TFIIB in transcription complex. 

 Bubble collapse define pol II promoter clearance transition.



Transcription factor TFIIF is not required for initiation by RNAPII, but it is essential to stabilise transcription factor TFIIB in early elongation complexes by Cabart et al
TFIIF is not required to initiate transcription , but it is essential for effective recruitment and retention of TFIIB.  PICs that lack TFIIF are fully functional in first-bond formation and promoter clearance 

Signification release of TFIIB occurs as nascent RNA is extended from 12 to 13 nt.

The activity of COOH-terminal domain phosphatase is regulated by a docking site on RNAPII and by the general transcription factors IIF and IIB by Chambers et al
Each cycle of transcription seems to be associated with reversible phosphorylation of repetitive COOH-terminal domain of largest RNAPII subunit. RNAPII dephosphorylation by CTD phosphatase is important.

Chambers et al suggest that RNAP II has a docking site for CTD phosphatase that is essential in dephosphorylation and is district from CTD.

CTD phosphatase was examined in presence of general TFs. TFIIF simulates CTD phosphatase activity 5fold. To identify TFs that might influence CTD phosphatase activity, purified or recombinant TFs were tested in a CTD phosphatase assay.  TFIIF does not stimulate dephosphorylation of RNAP IIO in absence of CTDF phosphatase. In lowe amounts of CTD phosphatase, TFIIF stimulates 5 fold.

TFIIB inhibits stimulatory activity of TFIIF on CTD phosphatase but has no effect on CTD phosphatase activity without TFIIF.

COOH-terminal domain of Rap74 is sufficient to stimulate CTD phosphatase.



TFIIF stimulates CTD phosphatase activity. CTD phosphatase was assayed in the presence of purified transcription factors as described under “Experimental Procedures.” Panel A: lane 1, no CTD phosphatase; lanes 2-8, contain CTD phosphatase; lane 2, no transcription factor; lane 3, 1.4 pmol of TFIIB; lane 4, 1.6 pmol of TBP; lane 5, 0.5 pmol of TFIIE; lane 6, 0.4 pmol of TFIIF; lane 7, 0.5 μg of TFIIH; and lane 8, 1 μg of TFIIJ. The amount of the transcription factors chosen was empirically determined as that being optimal for a reconstituted transcription reaction. Panel B, all lanes contain CTD phosphatase; lane 1, no TFIIF; lanes 2-6, 0.5, 1.3, 2.6, 5.2, and 13 fmol of TFIIF, respectively. Panel C, the CTD phosphatase activity from panel B was quantitated on a PhosphorImager and the photo-stimulated luminescence values plotted versus the TFIIF/RNAP IIO molar ratio. The assay contained 3 fmol of RNAP IIO. For simplicity, moles of TFIIF were calculated assuming a dimeric structure.
TFIIH: From transcription to clinic by Egly 
TFIIH has 10 or 11 subunits. One is XPD, a helicase in which a mutation gives rise to another form of XP. Another is cdk7. It phosphorylates CTD of the largest subunit of RNAPII.

TFIID: a 3D picture
Electron microscopy shows that TFIIH is organised into a ringlike structure with a hole. The hole can accommodate a ds DNA molecule 

Immunolabelling revealed quaternary organisation. cdk7 kinase is localised in protruding domain.  p44 is in ring structure and is flanked by XPB and XPD helicases.



XPB, helicase of transcription
Once PIC is formed, promoter opening at start site occurs in ATP dependent manner.  This allows further reading of coding strand by RNAPII.  This is due to ATP-dependent XPB helicase.

Mutating ATP binding site of XPB abolishes its function in both transcription and DNA repair.

Secondary role of XPD: opening and anchoring
Mammalian TFIIH contains core TFIIH which consists of 5 subunits (XPB, p62, p52, p44 and p34), and CAK composed of cdk7, cyclin H and MAT1.

XPD helicase subunit is believed to bridge CAK to core TFIIH. XPD allows anchoring of CAK to core TFIIH through interaction with p44. On interaction with p44, XPD helicase activity increases.

Phosphorylation by CAK is essential
CTD of largest subunit of RNAPII contains several repeats of s serine/threonin-0rich heptapeptide. it is an ideal substrate for ser/thr kinases. CTD phosphorylation/dephosphorylation follows certain steps of transcription. RNAPII engaged in elongation is hyperphosphorylated. Whereas initiation requires hypophosphorylated RNAPII.

Mutations in cdk7 or MAT1 decrease CTD phosphorylation and basal transcription 

CAK stimulates synthesis of first phoshodiester bond.



A history of TFIIH by Egly
ATPase activity of XPB is required to open DNA around TSS. ATP-dependent rotation of TFIIH downstream of a fixed RNAPII-promoter complex promotes DNA melting. Cdk7 of CAK phosphorylates CTD of Rpb1 of RNAPII.  CTD contains multiple TSPTSPS aa repeats. This enables switching between phosphylations of either Serine 2, 5 or 7 at different stages of transcription.

Cdk7 implements serine 5 phosphorylation of CTD during transcription initiation.  It is eilminated by Rtr1 phosphatase during elongation.

Mechanism of ATP-dependent promoter melting by transcription factor IIH by Kim et al
Models. (A) Promoter melting. In the presence of ATP, the IIH ERCC3 subunit rotates the DNA segment downstream of the transcription-bubble region relative to rotationally fixed upstream interactions, inducing melting of the transcription-bubble region. RNAPII is in dark blue, with the positions of the RNAPII RPB1, RPB2, and RPB5 subunits indicated; TBP, IIB, and IIF are in light blue; and IIH ERCC3 is indicated by an open red rectangle. Promoter DNA is drawn with upstream DNA at left, transcription-bubble region at center, and downstream DNA at right; the DNA segment contacted by the IIH ERCC3 is in red. ATP-dependent changes are highlighted in yellow. ADP, adenosine diphosphate; pi, inorganic phosphate. (B) Promoter escape. IIH function in promoter escape involves stimulating escape by transcription elongation complexes stalled after synthesis of 10 to 17 nt of RNA (10–12). IIH translocates with RNAPII during synthesis of the first 10 to 17 nt of RNA (23), and thus ERCC3 interacts with the DNA segment downstream of the transcription bubble in the stalled elongation complex [e.g., positions +16 to +42 for an elongation complex stalled at +13 (44); left]. In the presence of ATP, ERCC3 rotates the DNA segment downstream of the transcription bubble relative to the rotationally fixed upstream interactions, facilitating downstream extension of the transcription bubble and/or stabilization of the transcription bubble (right).

1) Entry of IIE and IIH into complex subtly alters protein-DNA interactions.

2) IIE makes extensive interactions with promoter DNA. IIE interacts with DNA in and immeditaly downstream of transcription bubble.

3) IIH makes extensive interactions with promoter DNA. Only ERCC3 subunit interacts with promoter. IIH interacts with DNA only downstream of bubble.

To detmine if IIH interactions with bubble in TCC in presence of ATP (when bubble is ss) crosslinking analysis.

1) Adding ATP leads to Rpb1-CTD pohspohrylation

2) Adding ATP does not alter protein-DNA interactions upstream of bubble

3) Adding ATP induces changes in protein-DNA inteactions in bubble

4) Adding ATP induces changes in IIH-DNA interactions downstream of bubble. IIH-DNA interactions are made only by ERCC3 subunit of IIH. They occur only downstream of bubble. They are made only with ds DNA in absence of ATP.

IIH does not interact with DNA in bubble region. This rules out for this promoter that IIH functions in promoter melting through conventional DNA-helicase mechanism.

That ERCC3 interacts with DNA downstream of bubble and adding ATP does not alter protein-DNA interactions upstream of bubble but alters interactions in and downstream of bubble suggests an alternative model for IIH function in promoter melting.

In the model, TCC in absence of ATP, sequence specific protein-DNA interactions by TBP and possibly IIF rotatationally fix promoter DNA upstream of bubble.

ERCC3  interacts with DNA downstream of ubble. On adding ATP, ERCC3 rotates DNA segment downstream of bubble by one turn relative to fixed upstream interactions. This induces melting of one turn of DNA between ERCC3 and fixed upstream interactions. Yields a ss transcription bubble.

ERCC3 acts as a wrench. It interacts with downstream DNA to generate torque. This nucleates formation of ubble, facilitates downstream extension of bubble and/or stabilises transcription.





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