Biochemistry 201
Biological Regulatory Mechanisms
January 26, 2015
C. Gross, Lecture 3
Eukaryotic transcriptional regulation
References
A few of the many insights from RNA polymerase structures
Cramer, P. (2002) Multisubunit RNA polymerases. Curr Opin Struct Biol 12:89-97.
Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the holo story. Curr Opin Struct Biol 13:31-9.
*Cramer, P. (2004) RNA polymerase II structure: from core to functional complexes. Curr Opin Genet Dev 14:218-26.
Review.
Wang, D. Bushnell DA, Westover KD, Kaplan, CD, Kornberg RD. Structural basis of transcription: role of the trigger loop
in substrate specificity and catalysis. Cell. 2006 Dec 1;127(5):941-54.
Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P.(2009) RNA polymerase II-TFIIB structure
and mechanism of transcription initiation. Nature. 462:323-30.
Chromosome conformation capture (CCC)
de Wit, E. and de Laat, W. (2012) A decade of 3C technologies: insights into nuclear organization. Genes Dev. 26: 11-24.
Mediator and Other Components
*Kornberg, R.D. (2005) Mediator and the mechanism of transcriptional activation. Trends in Biochemical Sciences
30:235-239.
Fan, X, Chou, DM, & Struhl, K. (2006). Activator-specific recruitment of Mediator in vivo. Nature Structural & Molecular
Biology, 13(2), 117-20.
Sikorski TW and Buratowski. (2009). The basal initiation machinery: Beyond the general transcription factors. Current
Opinion in Cell Biology. 21 344-351.
What do activators do?
Cosma, MP, Tanaka, T, & Nasmyth, K. (1999). Ordered recruitment of transcription and chromatin remodeling factors to a
cell cycle- and developmentally regulated promoter. Cell, 97(3), 299-311.
Bryant, GO, & Ptashne, M. (2003). Independent recruitment in vivo by Gal4 of two complexes required for transcription.
Molecular Cell, 11(5), 1301-9.
Bhaumik, S.R., Raha, T. Aiello, D.P., and Green, M.R. (2004) In vivo target of a transcriptional activator revealed by
fluorescence resonance energy transfer. Genes Dev 18: 333-343.
Vakoc, CR, Letting, DL, Gheldof, ... Blobel, GA (2005) Proximity among Distant Regulatory Elements at the B–Globin Locus
Requires GATA-1 and FOG-1. Molecular Cell 17:453-462
Fishburn, J., Mohibullah, N. and Hahn, S. (2005) Function of a eukaryotic transcription activator during the transcription
cycle. Molecular Cell 18:369-378.
Bulger M and Groudine M. Functional and Mechanistic Diversity of Distal Transcription Enhancers (2011). Cell 144:327-39
Role of the RNA Pol II CTD
*McCracken, S, Fong, N, Yankulov, K, et al. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing
to transcription. Nature, 385(6614), 357-61.
Tietjen,J. ……Ansari, A. Chemical-genomic dissection of the CTD code (2010) NMSB: 17: 1154-1162
Mayer, A. ….Cramer, P. Uniform transitions of the general Pol II transcription apparatus (2010) NMSB 17:1272-79
Buratowski, S (2009) progression through the RNA polymerase II CTD cycle ( Review). Mol Cell 36: 541-546
Chapman, R… Eick, D. Molecular evolution of the RNA polymerase CTD. Trends in Genetics (2008): Jun;24(6):289-96.
Epub 2008 May 9. Review.PMID: 18472177
Genome wide elongation technologies
Churchman LS, Weissman JS. (2011) Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature
469: 368-73. doi: 10.1038/nature09652. (Net-seq)
Kwak, H… and Lis, JT (2013) Precise maps of RNAP reveal how promoters direct initiation and pausing. Science 339: 950
(PRO-seq)
Elongation Control
BBA2013-- Issue 1874 devoted to reviews of transcription elongation
Yamaguchi, Shibata and Handa (2013):Transcriptionelongation factors DSIF and NELF: Promoter proximal Pausing and Beyond.
BBA 1829: 98-104
Note: this issue of BBA has many elongation reviews; most not too good.
Kwak and Lis (2013) Control of transcriptional Elongation Annual review Genetics 47: 483-508
Jonkers, Kwak and Lis (2014) Genome-wide dynamics of PolII elongation and its interplay with promoter proximal pausing, chromatin
and exons. E-life: 2014;3:e02407. DOI: 10.7554/eLife.02407
Veloso, Kirkconnell, magnuson, Biewen, Paulsen, Wilson and Mats Ljungman. (2014) Rate of elongation by RNA polymerase II is
associated with specific gene features and epigenetic modifications. Genome research 24:896–905
Core, Waterfall and Lis (2008) Nascent RNA Sequencing Reveals widespread pausing and divergent initiation at Human promoters.
Science 322: 1845-1848
Zhou Q, Li T, Price DH (2012) RNA polymerase II elongation control .Annu Rev Biochem. 2012;81:119-43.
Rougvie A and Lis JT (1988) The RNA Polymerase II Molecule at the 5’ end of the uninduced hsp70 gene of D. melangaster is
transcriptionally engaged. Cell 54: 795-804
Zobeck, KL….Lis Jt (2010) Recruitment timing and dynamics of transcription factors at the Hsp70 Loci in Living Cells Mol Cell
40 965-75
Nechaev S…..Adelman K. (2010) Global Analysis of short RNAs reveals widespread Promoter Proximal Stalling and Arrest of Pol II
in Drosophila Science 327: 335-38
Gilchrist, DA,……Adelman, K. (2010) Pausing of RNA Polymerase II Disrupts DNA specified Nucleosome Organization to enable
precise gene regulation. Cell 143: 540-51
Chen, Y.,….Handa, H.(2009) DSIF, the Paf1 complex and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II
elongation. Genees Dev 23: 2765 -77
Liu, Y……Hahn, S. (2009) Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of
the PAF complex. MCB 29: 4852-63
Wu, C-H,….Gilmour, D. ( 2003)NELF and DSIF cause promoter proximal pausing on the Hsp70 promoter in drosophila. Genes Dev
17: 1402-14
RNA polymerase elongation rate and splicing
Braberg, Jin, Moehle, Chan, Wang, Shales……Guthrie, Kaplan Krogan ( 2013) From Structure to Systems: High resolution
Quantitative genetic Analysis of RNA polymerase II. Cell 154, 775-788
Fong, Kim, Zhou, Jim Qiu, Saldi Diesner, Jones, Fu and Bentley (2014) Ore0mRNA splicing is facilitated by an optimal RNA
polymerase II elongation rate Genes and Dev 28: 2663-2676
Important Points
1. DNA looping allows proteins bound to distant sites on DNA to interact.
2. Transcription initiation at Pol II promoters on naked DNA templates in vitro requires the general transcription
factors in addition to RNA polymerase II.
3.. In vivo, transcription initiation also requires activators – proteins that bind directly to enhancers – as well as
Mediator and enzymes that modify chromatin structure.
4. At a typical eukaryotic promoter, activators guide the assembly of Mediator, the general transcription factors, RNA
polymerase and chromatin-modifying enzymes, often through weak, relatively non-specific interactions. There
appears to be no set order of assembly from one promoter to the next. Moreover, different promoters have different
requirements for these components.
5. The RNA polymerase CTD is a long series of 7-amino acid repeats. When transcription is initiated,
serine 5 of the repeat is phosphorylated by TFIIH. As elongation proceeds, serine 5 is gradually
dephosphorylated and serine 2 is gradually phosphorylated by enzymes carried along with the RNA
polymerase. This dynamic pattern of modification couples transcription to processing of the newlysynthesized RNA.
6. Promoter proximal pauses ( 20-50 nucleotides) are extensively used for regulation in eukaryotes.
7. Transcription takes place in a chromatin world and they exhibit a 2-way interaction
New challenges for transcription in eukaryotic cells
1. Three polymerases
2. Much more complex pattern of gene expression
Proliferation of trx factors
Regulation at a distance ( enhancers)
Combinatorial control
3. Transcription takes place in a chromatin world
Constant 2-way interplay between trx and chromatin
4. Complex processing of mRNA
PolII CTD serves as a platform for coordinating processes
PolII initiation factors and core promoter
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; coactivator
TFIIB
1
Recognizes BRE;Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment
1
~10
Binds TATA box; higher eukaryotes have multiple TBPs
Recognizes additional DNA sequences; Regulates TBP binding; Coactivator;
Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs
TFIID
TBP
TAFs
TFIIF
2
Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH;
enhances efficiency of pol II elongation
TFIIE
2
Recruits 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
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.
The first two steps of Eukaryotic transcription
On a supercoiled naked DNA template and in archae, TBP and TFB are
sufficient for formation of the pre-initiation complex (PIC), suggesting
that they are key to the mechanism of transcription initiation in
eukaryotes
TFB
TBP
Promoter
Many archae have a proliferation of TBPs and TFBs, suggesting that
they provide choice in promoters, akin to alternative s.
Transcription Initiation by RNA Pol II
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 complex
The GTF’s are not enough—what else is needed?
The concept of a co-activator
The GTFs are not sufficient to mediate activation:
Discovery and isolation of Mediator from Yeast
GTFs and RNA Pol II
Tx
1 unit
VP 16
GAL4
1 unit
crude lysate
10 units
4 years
mediator
50 units
Is Mediator Required for Transcription of all
Pol II -transcribed genes?
Control: ts subunit of Pol II
Rpb1
high T
Rpb1ts
Compare levels of all
mRNAs using microarrays
(37˚C for 45 min)
mRNAs decrease over time according to their half-lives.
A few mRNAs remain at some level (stable mRNAs).
Subunit of Mediator
Srb4ts
Same basic pattern
Subunit of TF II H
Kin28ts
Same basic pattern
Mediator is very large and has diverse roles
A)
PIC model from EM-study of polII (brown)-TFIIF
(light blue) and X-ray structure of TBP (white)
-IIB (yellow)--DNA; Arrow indicates direction of trx
B)
Model for PIC-mediator was produced by superimposing
an EM structure of Mediator-PolII on the PIC in A; head,
middle and tail regions shown
The Head region interacts with PolII-TFIIF complex; the mutants with general effects
on Trx are located in this region; the tail region interacts with activators; mutants have
more specific effects on transcription
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.
The TAFs in TFIID also serve as coactivators
Assembly of PIC in presence of mediator, activators
and chromatin remodelers
Many Paths to the PIC
Buratowski, 2009
The factors and assembly pathways used to form transcriptionally competent preinitiation complexes can be promoter
dependent. (1) TBP assembling onto promoter regions via TFIID leads to recruitment of the other basal initiation
factors, as outlined in the stepwise assembly pathway. In S. cerevisiae, this pathway is most often utilized at TATA-less
genes. At some mammalian promoters, histone H3K4 trimethylation helps to recruit the TFIID complex.
(2) Mediator bridges interactions between activators and the basal initiation machinery, and can stimulate basal
transcription as well. At some promoters Mediator can recruit TFIIH and TFIIE independently of RNApoII.
(3) TBP can also be brought to promoters by the SAGA complex. In S. cerevisiae, this pathway is most utilized at TATA
containing promoters. The Mot1 and NC2 complexes can repress this pathway by actively removing TBP from the TATA
element.
(4) Mot1 and NC2 can also have a positive role in transcription by removing non-productive TBP complexes from DNA,
thereby allowing functional PICs to form.
Genomic Level Snapshots of transcription factor
binding sites
ChIP
ChIP-ChIP
ChIP-seq
ChIP-exo
Bioinformatic approaches
Phylogenetic footprinting
Regulatory sequences expand in number and complexity
with increased complexity of the organism
~ 30-100 bp
~ 100s bp
Could be 50kB
or more
Are distant enhancers in proximity to the promoter?
Chromosome conformation capture-3C
3C
5C
1st example of 3C applied to enhancers: b globin locus:200kB
A hypothetical model of the active chromatin hub (ACH) is shown to illustrate the
3D nature of the ACH (not to scale), not the actual position of the elements
relative to each other in vivo. Red indicates the active regions (hypersensitive
sites and active genes) of the locus forming a hub of hyperaccessible chromatin
(ACH). The Inactive regions of the locus, having a more compact chromatin
structure are indicated in gray, with the inactive h1 and y genes in lighter gray.
Molecular Cell, Vol. 10, 1453–1465, December, 2002
Elongation control in eukaryotes
RNA polymerase II CTD
Heptad repeat unit
YSPTSPS
P
P
P
2
5
7
Plasmodium: 5
Yeast: 26
Mammals: 52
CTD is located adjacent to RNA exit channel
Palangat M et al. PNAS 2005;102:15036-15041
©2005 by National Academy of Sciences
Phosphorylation state of PolI CTD during transcription
2
Stage of transcription
Initiation
5
7
YSPTSPS
YSPTSPS
(Unphosphorylated)
Transition to elongation
Kinase/phosphatase
TF II H, Mediator
YSPTSPS
(Ser5)
P
pTEFb/Cdk9
Elongation
(Ser 2,5)
Further elongation
(Ser2)
Termination
(Unphosphorylated)
In S. cerevisiae:Cdk1 and Bur 1
YSPTSPS
P
P
Phosphatases (Rtr1(2?)
YSPTSPS
P
YSPTSPS
Phosphatases (Fcp1, ssu72)
A new step in transcription in higher
eukaryotes
NTPs
Prokaryotes
KB
R+P
RPc
Kf
RPo
Initiation
Abortive
Initiation
transition
Elongating
Complex
Elongation/termination
Higher Eukaryotes: same through abortive initiation, but new step before elongation
PIC ( does abortive initiation)
Promoter-proximal pause: +20 - +50
NELF: Negative elongation factor; DSIF: DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) sensitivity-inducing factor-Spt4+5
Potential roles for paused polymerase: antagonize nucleosome formation to keep promoter open;
synchronous activation; checkpoint
What is the major role of the Pol II CTD?
Mouse RNA Pol II
wt
52
 - amaR
CTD
5
50 hrs.
HeLa
cells
Introduce
CTD construct
examine
RNAs
Splicing, processing of 3’
end, termination
were all affected
 - amanitin
Nature 385: 357 (1997)
Specific Processes are connected to each Phosphorylated Form of the CTD
CTD Status
Transcription
Unphosphorylated
Serine 5P
RNA-Processing
Chromatin
mRNA capping
(capping enzyme)
H3K4 modification
Set1 complex
Activation
(mediator)
early termination
(ScN4E1 complex)
progression to elongation
(Cdk9 kinase via capping
enzyme); Bur1kinase)
Nucleosome mobility
Cdk9/bur1 for Spt5
Serine 2P/5P
Serine 2P
H3K36 methylation
(Set 2 )
late termination
(Rtt103)
polyadenylation
(Pcf11)
histone chaperone
Spt6
Histone marks: transcriptional regulators or by-products?
Approach: track residue specific modifications in living cells in a strain with a
GFP-glucocorticoid receptor and an inducible tandem array
Kimura doi:10.1038/nature13714
Results:
1. Preacetylated prior to addition of glucocorticoid
2. High acetylation correlated with rapid gene activation and rapid
transition to elongation
TECs are community organizers
The major steps in mRNA processing (trx, 5’ capping, polyA addition, splicing) all occur together on a transcript extruded from the
exit channel of RNAP although they can be reconstituted independently in vivo
Principles of “cotranscriptionality” to integrate nuclear metabolism
1.Permits coupling between different biogenesis steps; eg crosstalk; suspected when decreasing one step has effects on 2 nd; could
always be indirect
a. Landing pad—increase concentration of reactants—proteins involved in capping etc
b. Allosteary: guanosyl transferase of capping enzyme activated by interaction with phosphorylated CTD
c. Kinetic coupling—optimize timing
2. Impose order or control
a. Juxtaposition of proteins permits assembly, competitive interactions handoffs; often mutually exclusive PPis
b. directions emanating from phopshorylation state of CTD
3. A locator for nuclear machines –DNA repair, modification etc
Bentley: Cotranscriptionality Mol cell rev 2009
Kinetic coupling model for splicing
Approach: Measure splicing efficiency in a series of slow and fast RNAP;
Importantly double slow + fast returned splicing to normal
Caveats: not true for all splice sites; some reverse, some unaffected