SS 2009 – lecture 1Biological Sequence Analysis

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Transcription by Polymerase II in Drosophila

The 3 main phases of the transcription cycle are known as initiation, elongation

and termination. Each stage is subject to regulation.

During transcription initiation, a transcription-competent RNA polymerase complex

forms at the promoter and the DNA template is aligned in the active site of the

polymerase. The active site is where nucleotides are paired with the template and

are joined processively during elongation to produce the RNA transcript.

Termination of transcription involves release of the RNA transcript and the

dissociation of the transcription complex from the DNA template.

Regulation of transcription occurs at the level of RNA polymerase recruitment to

the promoter and at the level of elongation.

RNA polymerase II (Pol II) transcription elongation is divided into three distinct

stages: promoter escape, promoter-proximal pausing, and productive elongation.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Transcription by RNA polymerase II

Tamkun J. Nat. Gen. 39, 1421 (2007)

SS 2009 – lecture 1Biological Sequence Analysis

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C-terminal tail of RNA Polymerase II

This C-terminal domain distinguishes Pol II from the other two eukaryotic RNA

polymerases.

The number of repeats that exactly match the consensus sequence varies among

species.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

The C-terminal domain (CTD) of

the largest subunit of RNA

polymerase II (Pol II), Rpb1,

consists of tandem heptapeptide

repeats.

SS 2009 – lecture 1Biological Sequence Analysis

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Modifications of C-terminal tail of Pol II

E.g. CDK8 phosphorylates the CTD at Ser5.

Peptidyl-prolyl isomerases (e.g. yeast Ess1 and mammalian PIN1) can also alter

the conformation of the CTD, and regulate CTD phosphorylation and the binding of

other protein factors to Pol II.

Modification of the CTD is important for the coordination of transcription events.

Different modification states of the CTD are characteristic of different

transcriptional stages.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

The CTD can be modified by

phosphorylation (mostly at Ser2

and Ser5), glycosylation, and

cis/trans isomerization of prolines.

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Role of C-terminal tail of Pol II during elongation

During the transition from transcription initiation to elongation, Pol II changes from

a hypophosphorylated form to a hyperphosphorylated form.

Level of Ser5 phosphorylation peaks early in the transcription cycle and remains

constant or decreases towards the 3‘ end of the gene.

By contrast, Ser2 phosphorylation predominates in the body and towards the 3‘

end of the gene.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Transcription initiation

Before transcription initiation, a pre-initiation complex (PIC) forms at the

promoter, consisting of Pol and several general transcription factors (GTFs).

The GTFs position Pol II near the transcription-start site (TSS) and dictate the

precise location of transcription initiation.

The general transcription factor TFIIH is needed for the structural remodelling of

the PIC.

11-15 base pairs around the TSS are unwound to form an ‘open complex‘ that

allows the single-stranded DNA template to enter the active site of Pol II.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Promoter escape: elongation stage 1

Productively elongating Pol II can transcribe the full length of a gene in a highly

processive manner without dissociating from the template DNA or releasing the

nascent RNA product.

This is possible after promoter escape during which the polymerase breaks its

contacts with promoter-sequence elements and at least some promoter-bound

factors and simultaneously tightens its grip on the nascent RNA.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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a The unwinding of promoter DNA to create a transcription bubble begins at

a fixed position, ~20 base pairs downstream from the binding site of the TATA-

box-binding protein (TBP).

The upstream bubble edge (vertical dashed line) remains fixed until the

completion of promoter escape, whereas the downstream edge expands

together with transcription.

The initially transcribing complex (ITC) cycles through several rounds of

abortive initiation, releasing large amounts of 2–3-nucleotide-long RNA

transcripts (red).

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

Promoter escape – formation of an early elongation complex

SS 2009 – lecture 1Biological Sequence Analysis

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Escape commitment

b After synthesis of the first 4 nucleotides, the B-finger of TFIIB (orange) and a

switch domain (dark blue oval) of Pol II (large blue oval) stabilize the short RNA,

reducing abortive initiation.

This transition to a metastable transcription complex is termed escape

commitment.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Action of Polymerase II in Drosophila

c After 5 nucleotides are added, the nascent RNA collides with the B-finger of

TFIIB, inducing stress within the ITC.

This can cause increased abortive initiation, strong pausing, or transcript

slippage, if the nucleotides at the 3′ end of the RNA–DNA hybrid interact

weakly, and probably contributes to the rate-limiting step of promoter escape.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Promoter escape

d Stress from the growing transcription bubble and the production of a 7-

nucleotide-long RNA trigger collapse of the transcription bubble, providing the

energy to remodel the transcription complex.

The B-finger is ejected from the RNA-exit tunnel and TFIIB is released from the

transcription complex. The RNA–DNA hybrid is at its full length of 8–9 base

pairs and can make contacts with protein loops near the RNA-exit tunnel.

Abortive initiation ceases, as does the need for ATP hydrolysis, and transcript

slippage is markedly reduced, all indicating that the transcription complex has

changed into an early elongation complex (EEC).

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Transitioning to the pause region

Following promoter escape, the RNA remains stably bound in the transcription

complex, but has a tendency to undergo transcript slippage, backtracking and

arrest until about +30.

This phase is often accompanied by transcriptional pausing near the promoter.

Progress depends on stimulation by appropriate signals.

Consequently, this stage serves as a checkpoint for regulation.

The details how this step is regulated are subject of very active current research.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Zeitlinger et al. Nat. Genet. 39, 1512 (2007)

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Pol II binding profiles

Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007)

ChIP-chip assays were carried out with 2–4 h Toll10b embryos using antibodies

that recognize both the initiating and the elongating forms of Pol II.

y axis: enrichment ratios of Pol II.

(a–d) Binding patterns across genes that are repressed in Toll10b embryos. All

four genes show high Pol II signals near the transcription start sites. At some

genes, such as tup (a), Pol II is tightly restricted to this region, whereas at other

genes, including sog (c) and brk (d), Pol II is also detected at lower signals

throughout the transcription unit.

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Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007)

(e,f) Pol II is uniformly distributed across the transcription units of genes that are

actively transcribed. The stumps gene (e) is specifically activated in mesodermal

precursor cells, whereas RpL3 (f) is a highly expressed ribosomal gene.

(g,h) No Pol II binding is found at many genes that are inactive during

embryogenesis. The eyeless (ey) gene (g) is expressed during eye development

at larval stages but not in the early embryo. Likewise, the torso (tor) gene (h) is

active only during oogenesis but not in the early embryo.

SS 2009 – lecture 1Biological Sequence Analysis

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Promoter-proximal pausing

a TFIIH-mediated phosphorylation of Ser5 of the carboxy-terminal domain (CTD) of

RNA polymerase II (Pol II) occurs on pre-initiation complex formation or before

promoter-proximal pausing.

DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF)

probably facilitate Pol II pausing in the promoter-proximal region, and TFIIS also

associates with the paused polymerase.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

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Promoter-proximal pausing

b Positive transcription-elongation factor-b (P-TEFb)-mediated phosphorylation of

DSIF, NELF and Ser2 of the Pol II CTD stimulates productive elongation, and the

capping enzyme might contribute to this process by counteracting the negative

effects of DSIF and NELF 2. TFIIS facilitates efficient release of Pol II from the

pause site by aiding the escape of backtracked transcription complexes. NELF

dissociates from the transcription complex and DSIF, TFIIS and P-TEFb track with

Pol II along the gene. TFIIF, eleven-nineteen lysine-rich in leukemia (ELL), and

elongin, which stimulate Pol II elongation activity, might also associate with the

elongation complex.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Hendrix et al. PNAS 105, 7762 (2008)

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Hendrix et al. PNAS 105, 7762 (2008)

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Hendrix et al. PNAS 105, 7762 (2008)

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Nucleosome remodelling enzymes

a The four main families of ATPdependent chromatin remodellers are ISWI,

SNF2, CHD, and INO80. They are all related by virtue of a shared and conserved

ATPase, but differ markedly in their accessory proteins. Chromatin remodellers

can reposition nucleosomes in trans by transferring them to a different

DNA molecule (for example, from DNA b to DNA a) or in cis by sliding them

upstream or downstream from their original location.

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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Action of Polymerase II in Drosophila

b Nucleosome disassembly/reassembly factors, such as FACT and Spt6, can

facilitate transcription through chromatin by the removal of one histone H2A–

H2B dimer from the nucleosome.

Reassembly of the nucleosome after Pol II transcription is important for

preventing aberrant transcription initiation from cryptic promoters (sites that

contain a TATA element and a proximal initiation site).

Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)

SS 2009 – lecture 1Biological Sequence Analysis

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The H2A.Z histone in Drosophila

Heterogeneity in nucleosome structure results from incorporation of variant

histone proteins into the nucleosome.

In contrast to the canonical histones, which are multicopy genes expressed during

S-phase of the cell cycle, variant histones are encoded by single copy genes that

differ in amino acid sequence from S-phase histones and whose expression is not

limited to S-phase. Variant histones allow specialization of nucleosome

structure for specific purposes.

H2A.F/Z is a family of H2A variants that are highly conserved across species and

substantially divergent from S-phase H2A in any given species. H2Av is a variant

of H2A.Z in Drosophila. H2A.F/Z typically constitutes 5–10% of total H2A proteins

in the chromatin of cells.

H2A.F/Z may play a role in transcriptional regulation since in Drosophila its

incorporation into chromatin during development is coincident with the start of

zygotic gene expression.

Leach et al. J. Biol. Chem. 275, 23267 (2000)

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Leach et al. J. Biol. Chem. 275, 23267 (2000)