Saturday, 22 December 2012

Transcription elongation

Direct obsevation of bp stepping by RNAP by Abbondanzieri
a, A power stroke model where translocation (delta, red) is driven by irreversible PPi release. b, A brownian ratchet model where reversible oscillation between pre- and post-translocated enzyme states can occur before NTP binding (blue). c, A brownian ratchet model where translocation and NTP binding can occur in either order. This model postulates the existence of a secondary NTP site to accommodate the possibility of nucleotide binding when the enzyme is in its pre-translocated state.
a, Cartoon of the dumbbell geometry with schematic force versus position curves (dark red) shown for both trap beams (not drawn to scale). A single, transcriptionally active molecule of RNAP (green) is attached to a bead (blue) held in trap Tweak (pink, right) and tethered via the upstream DNA (dark blue) to a larger bead held in trap Tstrong (pink, left). The right bead is maintained at a position near the peak of the force-extension curve of Tweak, where trap stiffness vanishes (white arrow), creating a force clamp (trap stiffness k = dF/dx). During elongation, the DNA tether lengthens and the beads move apart. Owing to the force clamp arrangement, only the right bead moves: displacement is measured for this bead. b, Power spectrum acquired for a stiffly trapped bead with external optics under air (red) or helium (blue). Inset: integrated noise spectra for air (red) and helium (blue) showing a tenfold reduction in power. c, Steps resolved for a stiffly trapped bead moved in 1-Å increments at 1 Hz. Data were median filtered with a 5-ms (pink) and 500-ms (black) window. d, Steps resolved for a bead–DNA–bead dumbbell held at 27 pN of tension, produced by moving Tstrong in 3.4-Å increments at 1 Hz and measuring the corresponding displacements in Tweak.
Transcriptional pause, arrest and termination sites for RNAPII in mammalian N- and C-myc genes
Use purified RNAPII elongation complexes assembled on oligo (dC)-tailed templates or promoter-initiated RNAPII elongation complexes. Determine precise 3' ends of transcripts produced during transcription in vitro at human c- and N-myc pause, arrest and termination sites.

Many positions of pol II pausing, arrest or termination occurred in short regions of related sequence shared between c- and N-myc templates. Genes showed 3 classes of sequence conservation near intrinsic pause, arrest or termination sites:

1) sites where arrest or termination occurred after synthesis of runs of uridines preceding transcript 3' end
2) sites downstream of potential RNA hairpins
3) sites after nt addition following either a U or C or following a combination of several prymidines near transcript 3- end.

Mechanism of c- and N-myc regulation at level of transcript elongation may be similar.

Poised polymerases: on your mark ... get set ... go! by Price
A study suggests that in stem cells RNAPII initiates on most genes but only a fraction enters into productive elongation.  Many Drosophila genes contain poised polymerases.  One of these genes is hsp70. The class of genes with poised polymerases is highly enriched for developmental control genes eg genes encodig homeodomain proteins and genes that respond to developmental or environmental cues.

P-TEFb activity regulates developmental processes
P-TEFb reactivates poised polymerases. In a model, gene is repressed if it is occupied by posied pol. Activated when polymerase makes P-TEFb dependent transition into productve elongation.  P-TEFb is recruited by TFs.

P-TEFb fused to a DNA-binding domain instilled enhancer properties to cognate DNA-binding site.

In mammals P-TEFb is controlled by reversible interaction with snRP that contains HEXIM1 or HEXIM2.
Figure 2. Model for Elongation Control-Mediated Repression and ActivationThe repressed transcriptional state is characterized by the inactivation of P-TEFb by the 7SK snRNP and by the poised polymerase under the control of negative factors NELF, DSIF, and a currently unidentified factor(s) denoted with a question mark. In the activated state P-TEFb is released from the 7SK snRNA and productive elongation occurs through the aid of TFIIF. Phosphorylation of Ser2 and Ser5 (S2 and S5) in the heptapeptide repeat of the carboxyl terminal domain (CTD) of the large subunit of RNAP II, and phosphorylation of DSIF are indicated by red circles.

Unified two-metal mechanism of RNA synthesis and degradation by RNAP
In DNA-dependent RNAP, RNA synthesis and degradation reactions are perofmed by same active centre. Active centre involves a symnmetrical pair of Mg2_ ions. They switch roles in synthesis and degradtion. One ion is retrained permanently. the other is recruited for each act of catalysis. Weakly bound Mg2+ is stabilised in active centre in different modes depending on type of reaction.


Binding site for incoming NTP is i+1 site. RNA active centre for RNA terminus is i site. When phosphodiester bond is formed, terminus is translocated from i+1 to i site.

Reaction and movement are reversible. Pyrophosphate stimulates RNA degradation with release of 3' terminal NTPs.  Before pyrophosphorolysis, 3' terminus should return into i+1 site. TEC exists in equillbrium between i and i+1 site.

RNAP active centre can hydrolyse phosphodiester bond. In backtracked complex, RNa is threaded through active centre yielding a protruding 3' terminus. 3' fragment can be removed by intrinsic endonuclease activity.  Pyrophosphate can stimulate RNA cleavage in backtracked complexes of RNAPII, releasing an RNA fragment with 2'triphosphate.

RNA polymerase II elongation through chromatin by Orphanides and Reinberg
Nucleosomes are compacted to form chromatin. It is inaccessible to DNA-binding proteins. Eukaryotic cells may have specialised proteins to help RNAPII pass through chromatin during transcription elongation.

Proteins that decompact chromatin structure
Transcriptionally active accesible regions are associated with loss of protein involved with high order chromatin structure. Histone H1 binds nucleosomes and promotes chromatin unfolding.  Histone tail acetlyation disrupts histone-
DNa and inter-nucloeosmal interactions

RNAP meets nucleosome
DNA binding by activator proteins is prevented by chromatin packaging. Disrupting histone-DNA contacts overcomes repression.  Disruption of histone-DNA contacts byATP-dependent chromatin remodelling enzymes helps DNA binding. Allows DNA-binding proteins to compete with histones for DNA.

Histones remain associated with DNA of genes being transcribed. Felsenfeld et al found during elongation octamer of histones is transferred backwards on DNA fragment through transiently formed DNA loop. However RNAPII is stopped by nucleosomes, with strong pol-pausing sites in nucleosomes. These induce natural pausing.  Elnogation factors that accelerate elongation on free DNA cannot overcome this chromatin block  RNAPs recruit cellular factors which disrupt chromatin structure.

Helping RNA to elongate through chromatin
RNAPII in a cell travels at 25 nt per second. This rate can only be achieved on free DNA templates. Chromatin remodelling by SWI/SWF complex can promote RNAPII elongation through a nucleosome, by disrupting histone-DNA interactions.

Chromatin-associated HMG14 protein, found in chromatin of active genes, can slightly enhance RNAPII elongation through chromatin.

FACT complex can facilitate RNAPII elongation through nucleosomes. FACT interacts specifically with histones H2A and H2B. covalent crosslinking of histones in a nucleosome, to prevent removal of histones, abrogates FACT activity.  FACT may disrupt nucleosomes during RNAPII elongation by binding and removing histones H2A and H2B.  Yeast strains with mutant histone H4 (which later interaction of H2A and H2B with other histones) show same phenotypes as strains with mutations in Spt16 subunit of FACT.  Chromatin that contains transcribed sequences is deficient in H2A and H2B. It is preferentially bound by RNAPII.

Spt4, Spt5 and Spt6 proteins are implicated in relieving chromatin block to transcription.  Yeast with mutations in genes encoding these proteins have phenotypes consistent with defects in transcription elongation. Share many phenotypes with strains containing mutations in Sp16 subunit of FACT and in histones.

Human complex of Spt4 and Spt5 proteins bind RNAPII. Modulste its elongation on naked DNA templates in vitro. This complex can promote RNAPII elongation through chromatin templates in vitro. Spt6 can bind histones and alter chromatin structure in vitro.

Hitching a ride on RNAPII
Factors must be targeted to downstream region to facilitate elongation. It may ride on polymerase as it gtravels.  Must recognise and bind pols that are elongating, not free pols in nuclesu or at gene promoters. Tag that distringuishes a elongated pol may by phosphoylation of CTD tail.

PCAF HAT binds spec to phosphorylatd elongating RNAPII.

Svejstrup isolated an elongator, that associated only with phoshorylated elongation form of RNAPII. Contains Elp3 subunit with HAT activity.

Transcription elongation and histone acetylation
Maintaing histones in acetylated state requires constant transcription. State of histone tail acetylation is dynamic equilibrium determined by activities of HAT bound to elongating RNAPII and HDACs.
When RNAPII traffic along a gene is decreased, (governed by promoter signals), equilbrium shifts in favour of HDACs. Loss of acetylation may cause rapid conversion of chromatin to repressed conformation.



The RNA polymerase approaches the nucleosome and begins to synthesize a transcript (1; the DNA present in the nucleosome is shaded black). Transcription of the first 25 bp of DNA in the nucleosome is rapid and results in the displacement of DNA from the central histone octamer (2). The DNA segment behind the polymerase binds to the freshly exposed surface of the histone octamer, forming a DNA loop (3). Further polymerase elongation is hindered by this tight loop, which prevents the polymerase from rotating around the DNA as it reads the nucleotide sequence. The DNA behind the polymerase then transiently dissociates from the histone octamer, breaking the loop and allowing further elongation. This results in progression of the loop further into the nucleosome (4). This cycle of loop formation and breaking is repeated until the polymerase passes completely through the nucleosome (5; for details, see ref. 14). This process results in the transfer of the entire octamer of histone proteins backward on the same DNA segment. Figure adapted with permission from ref. 48.
Is a pioneer polymerase required?
2 models which differ in extent of chromatin decompaction after binding of activators to promoters are possible.

First model: activators recruit chromatin-modifying activities. Causes decompaction of chromatin surrounding activator binding sites and only partial decompaction elsewhere in gene. Elongating RNAPII faces a compacted chromatin template.

RNAPII must penetrate and unpackage repressive chromatin fibre. to facilitate this, first pol to transcribe a gene might be a specialised pioneer polymerase with additional tools to break down higher order chromatin structure  HATs that travel with subsequent elongating pols maintain chromatin in accessibly conformation.

2nd model: activators promote decompaction of chromatin over whole gene. Elongating RNAPII finds partially decompacted nucleosomes in its path.




One way in which chromatin-modifying activities can gain access to the whole transcribed region is by binding to RNAP II and travelling with it during elongation. Binding of activator proteins to the promoter results in recruitment of chromatin-modifying activities and local chromatin disruption (1). With the assistance of the general transcription factors, RNAP II then binds to the promoter region (2). Shortly after the initiation of transcription, the C-terminal domain (CTD) of RNAP II is hyperphosphorylated (3). RNAP II with a hyperphosphorylated CTD is recognized and bound by chromatin-modifying factors such as PCAF and/or elongator (4). The chromatin-modifying activities then travel with elongating RNAP II, leading to the propagation of chromatin disruption (5). This mechanism would enable the chromatin-modifying activities to gain access to the entire transcribed region and would result in the propagation of chromatin disruption through the entire length of the gene. For illustrative purposes only, the template ahead of the polymerase is shown here as a compacted chromatin fibre

Histones face the FACT by Svejstrup
Spt6 and Spt16 encode chromatin elongation factors.  Kaplan, Laprade and Winston show that an spt6 mutation impairs chromatin integrity in active genes. Mutant causes chromatin from a transcriptionlly active gene to be hypertensive to microccal nuclease. New TSSs also appear.

Belotsekovskaya, Reinberg et al show that FACT (comprising spt16 and pob3 gene products in yeast) promotes transcription-dependent nucleosome alterations. IT facilitates  assembly of histone proteins into nucleosome even in absence of RNAPII.

FACT is associated with actively transcribed RANPII genes on Drosophila polytene chromosome. RAPII can disassemble nucleosomes during transcription.

FACT removes one of 2 histone H2A/H2B dimers during RNAPII transcription through a nucleosome core particle.

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