1.Transcription initiation and its Regulation
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) and TADs
de Wit, E. and de Laat, W. (2012) A decade of 3C technologies: insights into nuclear organization. Genes Dev. 26: 11-24.
Merkenschlager M, Nora EP.CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation. (2016)
Annu Rev Genomics Hum Genet. 2016 Aug 31;17:17-43. doi: 10.1146/annurev-genom-083115-022339.
Gibcus JH, Dekker J.The hierarchy of the 3D genome. Mol Cell. 2013 Mar 7;49(5):773-82. doi: 10.1016/j.molcel.2013.02.011. Review. PMID: 23473598
Le TB, Laub MT. Transcription rate and transcript length drive formation of chromosomal interaction domain boundaries.
EMBO J. 2016 Jul 15;35(14):1582-95. doi: 10.15252/embj.201593561. PMID: 27288403
Le TB, Imakaev MV, Mirny LA, Laub MT.High-resolution mapping of the spatial organization of a bacterial chromosome.
Science. 2013 Nov 8;342(6159):731-4. doi: 10.1126/science.1242059. PMID: 24158908
Mediator and Other Components
Flanagan PM, Kelleher RJ 3rd, Sayre MH, Tschochner H, Kornberg RD (1991). A mediator required for activation of RNA polymerase II transcription in vitro Nature 350:436-8.
Allen BL, Taatjes DJ. The Mediator complex: a central integrator of transcription. Nat Rev Mol Cell Biol. 2015 Mar;16(3):155-66. doi: 10.1038/nrm3951.
Plaschka C, Nozawa K, Cramer P. Mediator Architecture and RNA Polymerase II Interaction. J Mol Biol. 2016 Jun 19;428(12):2569-74. doi: 10.1016/j.jmb.2016.01.028.
.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.
3.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
Basehoar AD, Zanton SJ, Pugh BF.(2004). Identification and distinct regulation of yeast TATA-box containing genes. Cell 116: 699-709
Levine M, Cattoglio C, Tjian R.Looping back to leap forward: transcription enters a new era. Cell. 2014 Mar 27;157(1):13-25. doi: 10.1016/j.cell.2014.02.009. Review. PMID: 24679523
Bothma JP, Garcia HG, Ng S, Perry MW, Gregor T, Levine. M Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo.
Elife. 2015 Aug 12;4. doi: 10.7554/eLife.07956. PMID: 26267217
Bothma JP, Garcia HG, Esposito E, Schlissel G, Gregor T, Levine M. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.
Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10598-603. doi: 10.1073/pnas.1410022111. PMID: 24994903
Fukaya T1, Lim B1, Levine M2 Enhancer Control of Transcriptional Bursting. Cell. 2016 Jul 14;166(2):358-68. doi: 10.1016/j.cell.2016.05.025. Epub 2016 Jun 9.
Bartman CR1, Hsu SC1, Hsiung CC1, Raj A2, Blobel GA3. Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping Mol Cell. 2016 Apr 21;62(2):237-47. doi: 10.1016/j.molcel.2016.03.007. Epub 2016 Apr 7.
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 : 771-8
*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
4.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)
Constant 2-way interplay between trx and chromatin
PolII CTD serves as a platform for coordinating processes
5.I. The basic eukaryotic transcription paradigm
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
7.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;accurately positions pol II
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)
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 complex
9.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.
10.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
General transcription factors
Chromatin and histone modification enzymes
11.The GTFs are not sufficient to mediate activation: Discovery and isolation of Mediator from Yeast
GTFs and RNA Pol II
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 with
Activators (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.
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 H3
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)
Yellow subunits: TAFs
(also part of TFIID)
TBP regulation (Spt3)
Assembly of PIC in presence of mediator, activators and chromatin remodelers
16.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: 699-709
17.III. PolII also has a unique structure (CTD) to coordinate transcription with other processes
RNA polymerase II CTD
Heptad repeat unit
. PNAS 102:15036-15041
CTD (800Å) is located adjacent to RNA exit channel
Mouse RNA Pol II
What is the major role of the Pol II CTD?
Splicing, processing of 3’ end, termination were all affected
Nature 385: 357 (1997)
20.Phosphorylation state of PolI CTD during transcription
TF II H, Mediator
In S. cerevisiae:Cdk1 and Bur 1
Phosphatases (Fcp1, ssu72)
Stage of transcription
Transition to elongation
see NSMB 23 : 771-8
Specific Processes are connected to each Phosphorylated Form of the CTD
CTD Status Transcription RNA-Processing Chromatin
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
Serine 2P/5P H3K36 methylation
(Set 2 )
Serine 2P late termination polyadenylation histone chaperone
(Rtt103) (Pcf11) Spt6
Heptad repeat unit
22.IV. Increasing complexity in metazoans
Spatial organization of genomes and
its role in gene regulation
New genomic and single cell microscopy approaches
Regulatory sequences expand in number and complexity with increased complexity of the organism
~ 30-100 bp
~ 100s bp
Could be 50kB
Chromosome conformation capture:
A method to probe nuclear organization
Spatial organization of the genome: Are distant enhancers in proximity to the promoter?
Are distant enhancers in proximity to the promoter?—
Chromosome Conformation Capture (CCC)
DNA contact maps
Methods have different names
depending on how the
contiguous DNA region is
In Hi-C, restriction enzyme
ends are filled in with biotin-
labeled nucleotides and
then pulled out with
26.1st 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-like
locus control region
27.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 49:773-82
Annu Rev Genomics Hum Genet. 2016 17:17-43
6. 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)
28.IV. Increasing complexity in metazoans
How 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.
1. 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.
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
At present, technology can detect the transcritpional output of enhance-promoter
interactions but is not yet able to directly visualize enhance-promoter interactions
Reporter System for examining how enhancers affect
2 color imaging uses: PP7 hairpin and PCP-tomato
Bursting output faithfully follows that of endogenous locus
31.Bursting frequencies correlate with enhancer strength
32.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.