i n Eukaryotes Lecture 2 12317 References A few of the many insights from RNA polymerase structures Cramer P 2002 Multisubunit RNA polymerases Curr Opin Struct Biol ID: 524488
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Slide1
Transcription initiation and its Regulation in Eukaryotes
Lecture #2
1/23/17Slide2
References
A few of the many insights from RNA polymerase structuresCramer, P. (2002)
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What do activators do?
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Role
of the RNA Pol II CTD
Zaboroska
, j;
egloff
s and murphy s. The
polII
CTD—new twists in the tail (2016) NSMB
23
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Tietjen
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Mayer
, A. ….Cramer, P. Uniform transitions of the general Pol II transcription apparatus (2010) NMSB
17:
1272-79Slide4
New challenges for transcription in eukaryotic cells
1. Three polymerases
2. Much more complex pattern of gene expression
3. Transcription takes place in a chromatin world
4. Complex processing of mRNA
Proliferation of trx factors
Regulation at a distance ( enhancers)
Combinatorial control
Constant 2-way interplay between trx and chromatin
PolII CTD serves as a platform for coordinating processesSlide5
I. The basic eukaryotic transcription paradigmSlide6
Three RNA polymerases
a. Pol I—ribosomal RNAs
*b. Pol II-protein coding genes + several small RNAs
c. Pol III-tRNAs, 5s rRNA, + several small RNAs
s
GreSlide7
Purification scheme for partially purified general transcription factors. Fractionation of
HeLa
nuclear extract (Panel A) and nuclear pellet (Panel B) by column chromatography and the molar concentrations of
KCl
used for
elutions
are indicated in the flow chart, except for the Phenyl Superose column where the molar concentrations of ammonium sulfate are shown. A thick horizontal (Panel A) or vertical (Panel B) line indicates that step elutions are used for protein fractionation, whereas a slant line represents a linear gradient used for fractionation. The purification scheme for pol II, starting from sonication of the nuclear pellet, followed by ammonium sulfate (AS) precipitation is shown in Panel B. (Figures are adapted from Flores et al., 1992 and from Ge et al., 1996)
NAME # OF SUBUNITS FUNCTION
TFIIA 3
Antirepressor; stabilizes TBP-TATA complex; coactivatorTFIIB 1 Recognizes BRE; Start site selection; stabilize TBP-TATA;accurately positions
pol II TFIID TBP 1 Binds TATA box; higher eukaryotes have multiple TBPs TAFs ~10 Recognizes additional DNA sequences; Regulates TBP binding; Coactivator; Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs TFIIF 2 Binds pol II; stabilizes pol II interaction with TBP and TFIIB; Recruits TFIIE and TFIIH; enhances efficiency of pol II elongation TFIIE 2 Recruits and regulates TFIIH; Facilitates forming initiation-competent pol II; promoter clearance TFIIH 9 ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoter
Pol II Initiation Factors (General transcription factors)Slide8
Transcription Initiation by RNA Pol
II on a naked DNA template
The stepwise assembly of the
Pol
II
preinitiation complex is shown here. Once assembled at the promoter,
Pol II leaves the preinitiation complex upon addition of the nucleotide precursors required for RNA synthesis and after phosphorylation of serine resides within the enzyme’s “tail”.
PIC = preinitiation complexSlide9
The Pol II promoter has many recognition regions
Positions of various DNA elements relative to the transcription start site (indicated by the arrow above the DNA). These elements are:
BRE (TFIIB recognition element); there is also a second BRE site downstream of TATA
TATA (TATA Box);
Inr (initiator element);
DPE (downstream promoter element);
DCE (downstream core element). MTE (motif ten element; not shown) is located just upstream of the DPE. Slide10
The GTF
’
s are not
sufficient to mediate activation—
what
else is needed?
The concept of a co-activator
II. Transcription Initiation by polII in vivo
Requirements:General transcription factorsActivatorsCo-activatorsChromatin and histone modification enzymesSlide11
The GTFs are not sufficient to mediate activation: Discovery and isolation of Mediator from Yeast
GTFs and RNA Pol II
Tx
1 unit
1 unit
10 units
crude lysate
4 years
50 units
mediator
VP 16
GAL4
Nature
350
:436-8
.Slide12
Mediator is very large and has diverse roles
Model of Mediator-
polII
initiation complex based on
cryo
-EM (9.7Å), lysine-lysine crosslinking, crystal structures of “core mediator” for both yeast and human, with largely similar results ( some mammalian specific extensions). Tail module may be positioned to interact withActivators (mutants in tail proteins have activation defects).
------------------------------------polII-silver, TBP-red, TFIIB-green, TFIIF-purple; Mediator: head—blue;middle—purple, tail—turquoise; dense parts have been crystallized.JMB 428:2569-Slide13
The TAFs in TFIID also serve as coactivators
TFIID—also an intimate chromatin connection:
TAF1 has HAT and double bromodomains;
TAF3 has PHD finger-recognizes Lys 4 of histone H3Slide14
SAGA is another important complex with multiple roles in transcription, including being a coactivator
The core of SAGA, containing the Taf substructure (Yellow), is surrounded by three domains responsible for distinct functions: activator binding (Tra-1), histone acetylation Gcn5), and TBP regulation (Spt3). This structural organization illustrates an underlying principle of modularity that may be extended to our understanding of other multifunctional transcription complexes.
Histone acetyl transferase (HAT)
(GCN5)
Yellow subunits: TAFs
(also part of TFIID)
TBP regulation (Spt3)
Activator binding—Tra-1Slide15
Assembly of PIC in presence of mediator, activators
and chromatin remodelersSlide16
Frequency of TATA-containing genes
Frequency of TATA-containing genes
The type of promoter can affect its regulation
About 20% of S.
cerevisiae
promoters have ~consensus TATA boxes ( dashed line in figures above).
Genes with consensus TATA boxes are highly enriched in genes that respond to stress ( left); are negatively affected by disabling a subunit of the SAGA complex (spt3), suggesting dependence on SAGA, and only marginally affected by mutation in a taf (taf1-2), suggesting weak dependence on TAFs (right)Cell
116: 699-709Slide17
III. PolII also has a unique structure (CTD) to coordinate transcription with other processesSlide18
RNA polymerase II CTD
YSPTSPS
P
P
P
Plasmodium: 5
Yeast: 26
Mammals: 52
Heptad repeat unit
2
5
7
.
PNAS
102:15036-15041
CTD (800Å)
is located adjacent to RNA exit channelSlide19
5
- ama
R
CTD
Mouse RNA Pol II
wt
52
What is the major role of the Pol II CTD?
examine RNAs
50 hrs.
HeLa
cells
Introduce
CTD construct
- amanitin
Splicing, processing of 3
’
end, termination
were
all affected
Nature 385: 357 (1997)Slide20
Phosphorylation state of PolI CTD during transcription
YSPTSPS
2
5
7
TF
II H, Mediator
pTEFb
/Cdk9
In
S. cerevisiae:Cdk1 and Bur 1
Phosphatases
(Rtr1(2?)
Phosphatases
(Fcp1, ssu72)
Stage of transcription
Kinase/phosphatase
Initiation
YSPTSPS
(
Unphosphorylated
)
YSPTSPS
P
Transition to elongation
(Ser5)
YSPTSPS
P
P
Elongation
(Ser 2,5)
YSPTSPS
P
Further elongation
(Ser2)
YSPTSPS
Termination
(Unphosphorylated)
see
NSMB
23
: 771-8Slide21
Specific Processes are connected to each Phosphorylated Form of the CTD
CTD Status
Transcription RNA-Processing Chromatin
Unphosphorylated
Activation
(
mediator)
Serine 5P early termination mRNA capping H3K4 modification
(ScN4E1 complex) (capping enzyme) Set1 complex progression to elongation Nucleosome mobility (Cdk9 kinase via capping Cdk9/bur1 for Spt5 enzyme
); Bur1kinase)
Serine 2P/5P H3K36 methylation (Set 2 )
Serine 2P late termination
polyadenylation
histone chaperone (Rtt103) (Pcf11) Spt6
YSPTSPS
Heptad repeat unitSlide22
IV. Increasing complexity in metazoansSpatial organization of genomes and
i
ts role in gene regulation
New genomic and single cell microscopy approachesSlide23
Regulatory sequences expand in number and complexity
with increased complexity of the organism
~ 30-100 bp
~ 100s bp
Could be 50kB
or more
Chromosome conformation
capture:
A method to probe nuclear organizationSpatial organization of the genome: Are distant enhancers in proximity to the promoter
? Slide24
Are distant enhancers in proximity to the promoter
?—
Chromosome Conformation Capture (CCC)
DNA contact maps
DiluteSlide25
Methods have different names
d
epending on how the
c
ontiguous DNA region is
analyzed
In Hi-C, restriction enzyme ends are filled in with biotin-labeled nucleotides and then pulled out with streptavidin beadsSlide26
1
st
example of 3C applied to enhancers:
b
globin locus:200kB
Molecular Cell, Vol. 10, 1453–1465, December,
2002
G & D 26: 11-24
The actively transcribed regions show close interactions with the enhancer-likelocus control regionSlide27
Some general features of spatial organization
Metazoan genomes appear to have widely spaced loci that interact with each other much more frequently than with random DNA. These are called “topologically associating domains (TADs) and are typically 100kB-1Mb in length. Many TADs contain both a promoter and their
enhancers
suggesting that they may be functional units.
2. Smaller TADs can be nested within larger TADs.
3. Neither existing
imaging technology nor Hi-C single cell technology currently have very good resolution at the level of an individual cell. Therefore, it is currently not clear how much TAD boundaries vary between cells. The current feeling, based on existing data is that boundaries are likely to fluctuate, allowing rewiring of contacts between enhancers and gene promoters.Remember that CCC captures the predominant conformations in a snapshot. Shome critical conformations, which could be shortlived intermediates may not be detected.
5. Boundaries are enriched in both transcription start sites and CTCF (CCCTC-Binding factor). CTCF is an architectural protein that can bind DNA strands together. As many CTCF sites are present within TADs, the rules of engagement are unclear. Mol Cell. 2013 49:773-82Annu Rev Genomics Hum Genet. 2016
17:17-436. Bacteria and yeast also have TADs. In bacteria, domains are called chromosome interaction domains (CIDs) and are on the order of 100 kd . Boundaries are established by highly expressed genes (hypothesis—transcription locally creates region of
under-wound DNA. CIDs may also be in higher order domains (Science. 2013 6159:731-4) Slide28
IV. Increasing complexity in metazoansHow do enhancers control gene expression?
Spatial organization of genomes
and
its role in gene regulation
Enhanced resolution imaging and higher quantum yield fluorescent probes are revolutionizing
our study of transcription in living cells. Slide29
Questions1. Which parameters of bursting do enhancers control?—predominantly fequency
2. What are the kinetics of bursting when 2 promoters are activated by the same enhancer?—somewhat coordinate
3. Insulators are functional units that disrupt enhancer-promoter communication. What is the effect of an insulator on bursting kinetics?—insulator decreases
fdrequency
of bursts but maintains coordination between promoters.
Overview: Visually examine the effects of genetically characterized enhancers-promoter interactions on transcription in live drosophila embryos at the maternal-zygotic transition when the 6000 nuclei are arranged as a monolayer. It has been established that in these systems, transcription occurs in bursts, characterized by : amplitude, duration, and freqeuncy
CaveatsAt present, technology can detect the transcritpional output of enhance-promoter interactions but is not yet able to directly visualize enhance-promoter interactionsSlide30
Endogenous locus
Reporter locus
Data output
Reporter System for examining
how
enhancers
affect
bursts2 color imaging uses: PP7 hairpin and PCP-tomatoBursting output faithfully follows that of endogenous locusSlide31
Bursting frequencies correlate with enhancer strengthSlide32
Coordination of transcriptional bursts from a single enhancer
The expectation from classical conceptions of enhancer/promoter
looping
is sequential bursting as the enhancer switches from one promoter to another. Instead, somewhat coordinate bursting.