Molecular and Structural Biology III
Core Promoter, RNA Pol II, GTFs,
Histone Modifications
Lecture 3
Dan Peet
[email protected]
Room 3.40 MLS Building
Gnatt et al. 2001, Science 292:1876-82
Today’s Lecture
• Core promoter/nucleosome positioning
• RNA Pol II
• General Transcription Factors
• Coactivators
• Histone modifications
• Histone Acetyl Transferases
Transcription
Proximal/Distal Promoter Core Promoter
INR
Nucleosome positioning
• Typical nucleosome positioning at a gene (ChIP sequencing)
• Two conserved nucleosome-free regions (NFRs), 5’ and 3’
Frequency
• Phasing tightest around 5’NFR
Jiang & Pugh, Nature Reviews Genetics 10:161-172 (2009)
Nucleosome positioning
• Typical nucleosome positioning at a gene
• Two conserved nucleosome-free regions (NFRs), 5’ and 3’
Jiang & Pugh, Nature Reviews Genetics 10:161-172 (2009)
• 5’ NFRs allow transcription, but not sufficient
• 5’ NFRs may allow assembly of PIC
• +1 nucleosome has strongest positioning, enriched for modifications
and variants that destabilise nucleosomes
• 3’ NFR where Pol II drops off – disassembly
Core Promoters
Transcription start.
Focussed promoters:
• single/small cluster of start sites
• widespread, but not in vertebrates
Dispersed promoters:
• multiple start sites over 50-100 bp
• common in vertebrates, rare in more ancient organisms
Long Vo ngoc et al. Genes Dev. 2017;31:1289-1301
Core Promoter
Transcription start.
BRE = TFIIB Recognition Element
TATA = TATA Box
Inr=Initiator
DPE=Downstream Promoter
Element
TCT = Polypyrimidine Inr
MTE = Motif Ten Element
XCPE=X Core Promoter Element
DTIE = Downstream Transcription
Initiation Element
Long Vo ngoc et al. Genes DCE = Downstream Core Element
Dev. 2017;31:1289-1301
• Elements vary with each gene (usually 0-3), also CpG islands
• Some core promoters lack all of these elements! (have atypical elements)
• Dispersed core promoters associated with CpG islands in vertebrates
• Dispersed core promoters commonly lack typical TATA, DPE & MTE motifs
• Could nucleosomes help position PIC and transcription start site?
Core Promoter
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Core Promoter
Contributions of motifs, initiators and trinucleotides
Motifs: the main sequence drivers of
transcription initiation signals (e.g. TATA Box)
- Directional and bidirectional
Initiators: only tune local base pair–level
location preference for transcription initiation
Trinucleotides: residual sequence
dependencies not captured by motifs and
initiators
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Contributions of motifs, initiators and trinucleotides
Motifs: the main sequence drivers of
transcription initiation signals (e.g. TATA Box)
- Directional and bidirectional
Initiators: only tune local base pair–level
location preference for transcription initiation
Trinucleotides: residual sequence
dependencies not captured by motifs and
initiators
Long Vo ngoc et al. Genes
Dev. 2017;31:1289-1301
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Contributions of motifs, initiators and trinucleotides
Motifs: the main sequence drivers of
transcription initiation signals (e.g. TATA Box)
- Directional and bidirectional
Initiators: only tune local base pair–level
location preference for transcription initiation
Trinucleotides: residual sequence
dependencies not captured by motifs and
initiators
Long Vo ngoc et al. Genes
Dev. 2017;31:1289-1301
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Contributions of motifs, initiators and trinucleotides
Motifs: the main sequence drivers of
transcription initiation signals (e.g. TATA Box)
- Directional and bidirectional
Initiators: only tune local base pair–level
location preference for transcription initiation
Trinucleotides: residual sequence
dependencies not captured by motifs and
initiators
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Motifs: the main sequence drivers of
transcription initiation signals (e.g. TATA Box)
- Directional and bidirectional
Initiators: only tune local base pair–level
location preference for transcription initiation
Trinucleotides: residual sequence
dependencies not captured by motifs and
initiators
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Core Promoter
Rate and position of initiation of transcription is controlled by contribution of all elements
Human promoters display very diverse motif combinations (so single motif in all promoters)
Most human promoters controlled by 3 motifs
Some motifs associated with tissue-specific expression (TATA) vs ubiquitous expression (YY1)
Activation of transcription is also influenced by distal enhancers
Dudnyk et al. Science 2024 Vol 384, Issue 6694
Transcription Regulation
KEY EVENTS FOR REGULATION:
1. State of eukaryotic DNA
2. Recruitment of Pol II & factors to promoter
3. Initiation of Transcription.
4. Promoter escape
5. Pausing
6. Elongation
7. Termination
8. Recycling
Fuda et al. (2009), Nature 461:186-192
RNA Polymerase II
• RNA Polymerase II responsible for transcribing all protein-encoding
nuclear genes.
• Yeast RNA polymerase II has been stringently purified.
• Consists of 12 subunits (total 513 kD). This is the core.
• Carboxy-terminal domain (CTD) on largest subunit, phosphorylated after
initiation.
• CTD plays key roles in transcription and mRNA processing.
• Purified yRNAPII inactive in vitro with naked DNA (unlike
prokaryotic RNAPII).
RNAP II
Crab-claw
structure Rudder
Unwinds RNA/DNA helix
Bridge deforms helix,
positions in active site
Closes down
Wall forces DNA up
at 90o angle
2 jaws
(Can’t see second jaw)
Klug, 2001, Science 292:1844-46
Question…
• Where are the histones in this structure?
• What happens to nucleosomes/histones during transcription?
?
RNAP II
• CTD consists of repeated heptad YSPTSPS (50
repeats in hRNAPII).
• All residues can be modified, some linked with
function
• Phosphorylated at Ser 5 during initiation
(releasing most GTFs).
• Phosphorylated at Ser 2 during elongation.
• CTD associated with RNA processing enzymes.
eg RNA capping, splicing and polyadenylation.
Figure 6-23 Molecular Biology of the Cell (© Garland Science 2008)
RNAP II
A. Mayer et al., 2010, Nature Struct. Molec. Biol. 17:1272-1279
RNAP II
Palancade and Bensaude 2003, Eur. J. Biochem. 270:3859-3870
RNA Polymerase II
• RNA Polymerase II responsible for transcribing all protein-encoding
nuclear genes.
• Yeast RNA polymerase II has been stringently purified.
• Consists of 12 subunits (total 513 kD). This is the core.
• Carboxy-terminal domain (CTD) on largest subunit, phosphorylated after
initiation.
• CTD plays key roles in transcription and mRNA processing.
• Purified yRNAPII inactive in vitro with naked DNA (unlike
prokaryotic RNAPII).
General Transcription Factors (GTFs)
• 5 additional factors required for transcription in vitro.
• Purified from HeLa cell nuclei.
• Named GTFs – general because they associate on all Pol II promoters.
• Also called General Initiation Factors and Basal Factors
• Together with Pol II they form the Pre-Initiation Complex (PIC).
General Transcription Factors (GTFs)
TFII = Transcription Factor for Polymerase II
• TFIID- TATA-Binding Protein (TBP) and 8-12 TATA Associated Factors
(TAFs). Binds core promoter elements, positions RNAPII.
• TFIIB- single protein
• TFIIF- 2 subunits
• TFIIE- 2 subunits
• TFIIH- 9 subunits, has two important enzyme activities (helicase and
kinase). Size is similar to RNAPII core.
(TFIIA- 3 subunits, not essential so not really a GTF, mediates the
magnitude of transcription)
General Transcription Factors (GTFs)
Overall role of GTFs?
➢ Correctly position RNAPII
➢ Permit DNA melting and promoter clearance once PIC is formed
➢ Target for activator interaction
IIF
IIE
IIB IIA IIH IID
TBP
TATA
box RNAPII
TFIID
• Binding of TBP to the TATA box through minor groove contacts.
• Induces a sharp bend in the DNA, opens up the structure.
TBP bound to DNA – “Saddle”
• Some of the TAFs interact
with/recruited by coactivators.
• Can bind to Core Promoter
Elements (Inr, DPE, etc)
• TAFII250 has kinase and acetylase
activity
Core Promoter
Transcription start.
BRE = TFIIB Recognition Element Inr=Initiator
MTE = Motif Ten Element DPE=Downstream Promoter Element
DCE = Downstream Core Element XCPE1=X Core Promoter Element 1
• TFIID recognises TATA, Inr, MTE and DPE
• TFIIB recognises BREu and BREd
TFIIH
• Plays crucial roles in:
• Transcription initiation
• Transcription elongation
• RNAPI-mediated transcription (rRNA) Australian 16/04/2022, Picture: Toby Zerna
• DNA repair
Australian 16/04/2022, Picture: Justin Benson-Cooper/The West Australian
AusPol.co 09/04/2022
• Cell Cycle regulation
Herald Suno 04/05/2022, Picture: Liam Kidston
• Contains 9 subunits.
Illawarra Mercury 21/04/2022,
Daily Mail 19/05/2022,
AusPol.co 17/05/2022,
Inside Story 31/01/2020, Picture: James Ross/AAP Image Daily Mail 20/01/2022,
SBS News 18/04/2022, Picture: James Ross/AAP Image
TFIIH
• Contains both helicase and kinase activity.
• Helicase activity required to open up/melt promoter DNA (mechanism
uncertain) in transcription and DNA repair. Australian 16/04/2022, Picture: Toby Zerna
AusPol.co 09/04/2022
Australian 16/04/2022, Picture: Justin Benson-Cooper/The West Australian
• Kinase activity responsible for phosphorylation of RNAPII CTD
at Ser 5 during initiation (Cdk7/Kin28). Herald Suno 04/05/2022, Picture: Liam Kidston
Illawarra Mercury 21/04/2022,
Daily Mail 19/05/2022,
AusPol.co 17/05/2022,
Inside Story 31/01/2020, Picture: James Ross/AAP Image Daily Mail 20/01/2022,
SBS News 18/04/2022, Picture: James Ross/AAP Image
How is PIC Assembled?
IIF
IIE
IIB IIA IIH IID
TBP
TATA
box RNAPII
PIC Assembly – Ordered Model
• Based on gel shift data and footprinting with purified
GTFs In Vitro
• BUT often quite different in vivo (in living cells)
So don’t memorise!
Something Missing!
TATA
YFG
box RNAPII
Activator/signal-regulated transcription factors bind DNA but do not
commonly alter DNA structure or directly bind the PIC
What physically alters the higher order structure of DNA?
What connects the activator/signal-regulated transcription factors and the
PIC, recruits the PIC?
How do they communicate?
Transcriptional Coactivators
COACTIVATORS:
• Diverse range of proteins, most in large complexes (some over 1 MDa).
• Some are components of the basal transcription machinery
ROLE OF COACTIVATORS:
1. Communicate with activators and GTFs/PIC.
2. Remodel and modify chromatin.
3. Recruit transcription machinery.
Transcriptional Coactivators
Three groups:
Modify chromatin structure.
1. Covalent histone modifiers.
2. ATP-dependent chromatin remodellers (non-covalent).
Recruit basal transcriptional machinery, and mediate communication
between activators and PIC
3. Mediator.
Transcriptional Coactivators
Three groups:
Modify chromatin structure.
1. Covalent histone modifiers.
2. ATP-dependent chromatin remodellers (non-covalent).
Recruit basal transcriptional machinery, and mediate communication
between activators and PIC
3. Mediator.
A comprehensive view of the PTM landscape in the human proteome.
N. Kitimura & J.J. Galligan, Biochem J (2023) 480: 1241–1265
At least 28 Different Types of Histone PTMs
Millam-Zambrano et al., Nature Reviews Genetics 116: https://doi.org/10.1038/s41576-022-00468-7 (2022)
Histone Modifications
Q. How do you identify novel post-translational modifications?
A. First - mass spectrometry (identify)
Second – develop specific antibodies for ChIP to show where and when
H1.2 H2A H2B H3 H4 Total
# sites 31 12 31 33 23 130
# novel 17 12 19 10 11 67
K-me 7 3 6 - 2 18
K-me2 - - - - 1 1
K-fo 1 - 2 1 - 4
K-ac 1 - - 1 - 2
R-me - 2 1 2 3 8
Y-oh 1 1 2 - 2 6
K-cr 7 4 8 6 3 28
Tan et al., Cell, Volume 146, Issue 6, 2011, 1016 - 1028
Key Histone Modifications
Khorasanizadeh et al., Cell 116: 259-272 (2004)
Further Reading
• Dudnyk et al. Sequence basis of transcription initiation in the human genome. Science 2024 Vol
384, Issue 6694. (Recent research paper on core promoter elements controlling transcription)
• Rimel JK, Taatjes DJ. The essential and multifunctional TFIIH complex. Protein Sci. 2018. doi:
10.1002/pro.3424. (Up-to-date review on the Eddie McGuire of transcription factors)
• Lee KK, Workman JL. Histone acetyltransferase complexes: one size doesn't fit all. Nat Rev
Mol Cell Biol. (2007) 8:284-95. (Great review on HATs – with a punny title I would be proud of)
• Kouzarides T. Chromatin modifications and their function. Cell. (2007) 128:693-705.
(A great recent review on all of the different histone modifications and what they might do).
• Li B, Carey M, Workman JL. The role of chromatin during transcription. Cell (2007) 128:707-19.
(Another very good review that includes a lot of information about chromatin related to
transcription, including histone modifications and much more)
• Srivastava R, Ahn SH. Modifications of RNA polymerase II CTD: Connections to the histone
code and cellular function. Biotechnol Adv. (2015) 33:856-72. (A review focussed on the CTD and
roles of multiple modifications in transcription).
• Millam-Zambrano et al., Histone post-translational modifications — cause and consequence of
genome function Nature Reviews Genetics 116: https://doi.org/10.1038/s41576-022-00468-7
(2022) (Very good recent review of histone PTMs)
