Transcript BioReg2014_Transciption_2

Control of Transcription Initiation
General References
Chapter 16 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M,
Losick, R. 547-587.
Ptashne, M. and Gann, A. (2002) Genes and Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Luscombe, N.M., Austin, S.E., Berman, H.M., Thornton, J.M. (2000) An overview of the structures of protein-DNA
complexes. Genome Biology 1(1): reviews001.1-001.37
Examples of Control Mechanisms
Alternative Sigma Factors
Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex 28 /FlgM reveals an
intact sigma factor in an inactive conformation. Molecular Cell 14:127-138.
Gruber, TM, Gross, CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev.
Microbiol 57:441-66
Increasing the Initial Binding of RNA Polymerase Holoenzyme to DNA
Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. (2004) Catabolite activator protein: DNA binding
and transcription activation. Curr Opin Struct Biol. 14:10-20.
Increasing the Rate of Isomerization of RNA Polymerase
*Dove, S.L., Huang, F.W., and Hochschild, A. (2000) Mechanism for a transcriptional activator that works at the
isomerization step. Proc Natl Acad Sci USA 97: 13215-13220.
Jain, D. Nickels, B.E., Sun, L., Hochschild, A., and Darst, S.A. (2004) Structure of a ternary transcription activation complex.
Mol Cell 13: 45-53.
Hawley and McClure (1982) Mechanism of Activation of Transcription from the l PRM promoter. JMB 157: 493-525
DNA looping
**Oehler, S., Eismann, E.R., Kramer, H. and Mueller-Hill, B. (1990) The three operators of the lac operon cooperate in repression.
EMBO 9:973-979.
Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331:981-989.
Dodd, I.B., Shearwin, K.E., Perkins, A.J., Burr, T., Hochschild, A., and Egan, J.B. (2004) Cooperativity in long-range gene regulation
by the l cI repressor. Genes Dev. 18:344-354.
*Choi, PJ, Cai,L, Frieda K and X. Sunney Xie (2008) A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell
Science 2008: 442-446. [DOI:10.1126/science.1161427]
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 cycleand 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
Important Points
1. Every step in transcription initiation can be regulated to increase or decrease the number of successful initiations
per time.
2. In E. coli, transcription initiation is controlled primarily by alternative  factors and by a large variety of other
sequence-specific DNA-binding proteins.
3. G=RTlnKD. This means that a net increase of 1.4 kcal/mole (the approximate contribution of an additional
hydrogen bond) increases binding affinity by 10-fold. Many examples of transcription activation in bacteria take
advantage of such weak interactions.
4. To activate transcription at a given promoter by increasing KB, the concentration of RNA polymerase in the cell
and its affinity for the promoter must be in the range so an increase in KB makes a difference. Likewise, to activate
transcription by increasing kf, the rate of isomerization must be slow enough so the increase makes a substantial
difference.
5. DNA looping allows proteins bound to distant sites on DNA to interact.
6. Transcription initiation at Pol II promoters on naked DNA templates in vitro requires the general transcription
factors in addition to RNA polymerase II.
7.. In vivo, transcription initiation also requires activators – proteins that bind directly to enhancers – as well as
Mediator and enzymes that modify chromatin structure.
8. 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.
Control of Transcription Initiation in Bacteria
Every step of transcription can be regulated
NTPs
KB
R+P
RPc
initial
binding
Kf
RPo
“isomerization”
Abortive
Initiation
Elongating
Complex
Gene regulation in E. coli: The Broad Perspective
• 4400 genes
• 300-350 sequence-specific DNA-binding proteins
• 7  factors
Alternative s are major control mechanism in bacteria
Regulation by repressors and activators
(alter reactivity of 70-holoenzyme)
A brief digression: How proteins recognize DNA
All 4 bp can be distinguished in the major groove
Common families of DNA
binding proteins
In vivo parameters for Sequence-Specific DNA binding proteins
KD ≈ 10-6 - 10-10M in vivo
In E. coli 1 copy/cell ≈ 10-9 M
If KD = 10-9M and things are simple:
10 copies/cell
90% occupied
100 copies/cell
99% occupied
I. Regulating transcription initiation at KB (initial binding) step
Negative control: repressors (e.g. l, Lac ); prevent RNAP binding
R
-35
-10
Positive control: activators ( e.g. CAP); facilitate RNAP binding with
favorable protein-protein contact
Favorable
contact
A
*
RNAP holo
-35
-10
Lac repressor and DNA looping
Lac ~ 1980
-35
-10
Lac operator
Lac 2000
O3
-90
O1
-35
-10
O2
+400
Oehler, 2000
O2
1/10
affinity of O1
O3
1/300 affinity of O1
What is the function of these weak operators?
The weak operators significantly enhance represssion
Oehler, 2000
Through DNA looping, Lac repressor binding to a “strong” operator (Om)
can be helped by binding to a “weak” operator (OA)
OK
Om
Oa
Better!
Om
A mutant Lac repressor that cannot form
tetramers is not helped by a weak site
MM
Theoretical consideration of effects of looping (2 operators)
Representative states of the binding of the repressor to one operator (top
panel) or to two operators (bottom panel). Om (main operator) binds
repressor more tightly than Oa (auxiliary operator). Transcription takes
place only in the states (i) and (iii), when Om is not occupied. The arrows
indicate the possible transitions between states. Note that with one
operator, a single unbinding event is enough for the repressor to
completely leave the neighborhood of the main operator. With two
operators, the repressor can escape from the neighborhood of the main
operator only if it unbinds sequentially both operators.
From: Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical
constraints on transcription regulation. J Mol Biol 331:981-989
.
I. Regulating transcription initiation at KB (initial binding) step
Positive control: activators ( e.g. CAP); facilitate RNAP binding with
favorable protein-protein contact
Favorable
contact
A
*
RNAP holo
-35
-10
∆ G = RT lnKD; if * nets 1.4 kcal/mol, KB goes up 10-fold
Activating by increasing KB is effective only if initial promoter
occupancy is low
If favorable contact nets 1.4Kcal/mole (KB goes up 10X) then:
a) If initial occupancy of promoter is low
RNAP
A *
RNAP
10% occupied
1% occupied
Transcription rate increases 10-fold
b) If initial occupancy of promoter is high
RNAP
99% occupied
A *
RNAP
99.9% occupied
Little or no effect on transcription rate
A case study of activation at KB: CAP at the lac operon:
How is CAP activated?
cAMP
Inactive CAP
high glucose
Active CAP
Regulates >100 genes positively or negatively
CAP at lac operon
CAP increases transcription ~40-fold; KB ; no effect on kf
Strategies to identify point of contact between CAP and RNAP
1. Isolate “positive control” (pc)
mutations in CAP. These mutant
proteins bind DNA normally but do
not activate transcription
M
M
2. “Label transfer” (in vitro) from
activator labeled near putative
“pc” site to RNAP
Activate X*; reduce S-S; X* is
transferred to nearest site;
determine location by protein
cleavage studies; X*
transferred to -CTD
3. Isolate CAP-non-responsive
mutations in -CTD
S-S-X*
RNAP
-35
-10
M
RNAP
-35
-10
Signal transduction cascade
Signal
Transcription factor
Genes controlled by transcription factor
Common network motifs in
transcriptional circuits
Using a feed-forward loop
to measure signal duration
Control of Transcription Initiation in Eukaryotes
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”.
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
Rpb1ts
high T
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 is a variant of ChIP. Cells are treated with formaldehyde to create DNA-protein-DNA
cross-links. (Formaldehyde reacts with the amino groups on proteins and nucleic acids to
form protein-protein and DNA protein covalent linkages). The DNA is then treated with a
restriction nuclease that produces cohesive ends. Prior to the ligation step, the DNA is
diluted so that the DNA ligase will join two different DNA fragments only if they are
cross-linked. Finally, the cross-links are reversed and the DNA is purified, so that the
ligated DNA molecules can be quantified by PCR.
3C reveals proximity of enhancers and promoters eg. colocalization in the ligated DNA product; eliminating
transcription of a gene eliminates colocalization of enhancer and promoter sequences ( eg. b-globin locus)
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
5C technique
5C applied to ES and NPC cells around key developmentally regulated genes to identify
topologically associated domains (TAD)
Cell 153, 1281–1295, June 6, 2013
Locus specific proteomics
Identify proteins ( mass spectrometry)
test their functional significance