Transcription
There are 9 steps in Transcription.
- Chromatin opening
- Pre-Initation Complex Assembly (PIC)
- Initation
- Promoter Escape
- Promoter-Proximal Pausing (PEC)
- Pause Release
- Productive Elongation (EC*)
- Termination
- Recycling
Fuda, Ardehali, & Lis Nature 2009
Chromatin Opening
PIC Assembly
Promoter Escape
During open complex formation, TFIIH core helicase activity unwinds DNA to form open complex, and TFIIB aids TSS selction and closed complex formation by interacting with TBP, DNA and Pol II.
TFIIB N-terminal B-finger is important for stablizing a 5nt RNA within Pol II active site. But it also blocks the RNA exit channel, thus continued elongation must evit the B-finger.
Saunders et al. (2006) Nature Reviews
Sainsbury et al. (2013) Nature
Transcription complex is now stable.
Promoter-Proximal Pausing
Promoter-Paused Elongation Complex(PEC): Pol II-DSIF-NELF
DSIF: DRB Sensitivity Inducing Factor
NELF: Negative ELongation Facotr
GAF, GTFs and core promoters also promote promoter proximal pausing.
NELF could block binding of other positive factors.
NELF binds the Pol II funnel and could restrict NTP diffusion, reducing substrate delivery to active site. (A tilted DNA–RNA hybrid impairs binding of an NTP substrate.) NELF restrains Pol II mobility required for pause release.
Vos et al. (2018) Nature 560:601
PRO-seq shows 2 classes of pausing: (20-60nt)
- Genes with focused polymerase peaks near the TSS. (TATA-box, GAGA and GAF enriched)
- Genes with dispersed polymerase peaks farther from the TSS.
Pausing is higher when promoter elements are in their consensus positions.
Kwak et al. (2013) Science 339:950
Pausing can lead to backtracking of Pol II (3' end of the RNA threads out into the secondary channel). And Severe backtracking can lead to arrest. TFIIS induces Pol II's intrinsic cleavage activity and puts a 3'-OH back into the active site. After this Pol II can resume transcription.
Kettenberger et al. (2003) Cell
Why Pause?
Main hypotheses for the fuctions of Pol II pausing:
1. Establishing permissive chromatin
After nucleosomes have been remodeled, paused Pol II helps to maintain the nucleosome-deprived structure by blocking nucleosome assembly over promoter sequences. Pausing would thus keep the promoter region accessible for activator and transcription factor binding.
Adelman & Lis (2012) Nat Rev Genet 13:720
2. Rapid and/or synchronous activation
Mike Levine group has shown TFs of the early embryo are expressed from genes that show high levels of paused Pol II. These TFs need to be turned on rapidly and synchronously in the early embryo to produce normal development. Substituting core promoters with lower pausing leads to non-synchronous expression of these TFs and abnormal development.
Pause Release
Pol II pause release is dependent on P-TEFb kinase. (Positive Transcription Elongation Facotr-b)
P-TEFb localizes to actively transcribed genes, phosphorylates the CTD Serine2 residues, Spt5 CTR domain and NELF. Inhibition of P-TEFb prevents Pol II from entering the gene body. (David Price's lab and John T. Lis's lab)
NELF is released by the phosphorylated complex.
Saunders (2006) Nat Rev Mol Cell Biol. 7: 557
Productive Elongation
Elongation Complex(EC*): Pol II-DSIF-PAF-SPT6
PAF can reverse NELF-stabilized Pol II pausing in vitro when active P-TEFb and ATp are present allowing transcription elongation and evicting NELF. Elongation is further stimulated when SPT6 is also present.
Vos et al (2018) Nature 560:607
Transcription through a nucleosome
Nucleosomes present a barrier to transcription
DNA needs to be unwrapped from nucleosome to allow Pol II to pass.
Transcription through a nucleosome can be split up in three nucleosomal regions: The entry, in which DNA partially unwraps, and DNA mostly interacts with histone tails and H2A/B; the central part, where DNA interacts tightly with core histone H3/4, and the exit region where interaction are primarily between H2A/B again.
TFIIF and/or TFIIS couldn't overcome the nucleosomal block to transcription. The group of Danny Reinberg searched the fators that FAcilitates Chromatin Transcription (FACT), they found Spt16 and SSRP1.
Orphanides et al., Cell (1998)
FACT & Spt6 disassemble and reassemble nucleosomes as Pol II passes. And Cryo-EM from Luger lab is beginning to provide molecular details of this process.
Belotserkovskaya & Reinberg Current Opinion in Genetics & Development (2004)
Elogation Rates
Exons reduce elogation rates (more exons, slower elogation). CpG content and H3K79me2 correlated with decreased and increased elogation rate respectively.