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.
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.
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.
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.