Transcript Access Slides

The Eukaryotic Gene Transcription Machinery:
Mechanism and Regulation
Zhen-Qiang Pan, Ph.D
March 24, 2005
Outlines:
1) An overview of the eukaryotic gene transcription
2) Mechanisms of transcription by Pol II
3) Action of Mediator
4) Enhancer-promoter communication during gene activation
Summary:
A common multi-protein machinery transcribes many thousands of genes coding for proteins in eukaryotes.
Recent structural studies have provided Information about the Pol II-based eukaryotic transcription
machinery and about Mediator, the complex involved in transcription regulation during initiation. We will
discuss the current model concerning the possible mechanisms of transcription initiation and regulation.
A model study will be presented concerning the order of recruitment of factors to the HNF-4 regulatory
regions upon the initial activation of the gene during enterocyte differentiation. The results provide
experimental evidence for the involvement of a dynamic process culminating in enhancer-promoter
communication during long-distance gene activation.
References:
Cramer P, Bushnell DA, Kornberg RD. 2001. Structural basis of transcription: RNA polymerase II at
2.8 angstrom resolution. Science 292(5523):1863-76.
Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD. 2001. Structural basis of transcription: an
RNA polymerase II elongation complex at 3.3 A resolution. Science 292(5523):1876-82.
Davis JA, Takagi Y, Kornberg RD, Asturias FA. 2002. Structure of the yeast RNA polymerase II
holoenzyme: Mediator conformation and polymerase interaction. Mol Cell 10(2):409-15.
Hatzis P, Talianidis I. 2002. Dynamics of enhancer-promoter communication during differentiation-induced
gene activation. Mol Cell 10(6):1467-77.
Asturias FJ. 2004. RNA polymerase II structure, and organization of the preinitiation complex.
Curr Opin Struct Biol. 14(2):121-9. Review.
An overview of the eukaryotic gene transcription
By Alberts
TBP
TATA
By Alberts
By Alberts
Mechanisms of transcription by Pol II
By Kornberg, (2001) Biol. Chem, 382, 1103-7
Structure of RNA polymerase II. Cutaway view, to reveal contents of active center cleft. Surface
representation of atomic model, with features colored as follows: clamp, orange; wall, blue; bridge helix, green;
active center Mg ion, pink; and remainder of polymerase, gray. A) Transcribing complex, with coding strand of
DNA in active center region in turquoise, and RNA in red (PDB 1I6H). B) RNA polymerase II – TFIIB complex,
with backbone model of TFIIB in yellow (PDB 1R5U).
By Kornberg, (2005) FEBS Letters, 579, 899-903
Structure of RNAPII and interaction of the enzyme with promoter DNA. This schematic representation
of the polymerase (shown in orange) emphasizes the way in which the clamp and wall domains restrict access
to the active site. Subunits Rpb4 and Rpb7 form a complex (shown in blue) that can dissociate from the core
enzyme, and might play a role in helping to determine the position of the clamp domain. The Rpb4–Rpb7 complex
may also be involved in interaction with newly synthesized RNA. The narrow configuration of the active site cleft
probably requires melting of the transcription start region for the template strand to reach the RNAPII active site
(indicated by the red dot).
Asturias FJ. 2004. Curr Opin Struct Biol. 14(2):121-9. Review.
RNA polymerase II transcription initiation complex. X-ray and electron microscope
structures (upper left) were assembled in a complete transcription initiation complex
(lower right).
By Kornberg, (2005) FEBS Letters, 579, 899-903
Action of Mediator
By Bjorklund & Gustafsson, (2004) Advance in Protein Chem., 67, 43-65
Tail
The yeast RNA Polymerase II holoenzyme revealed by electron microscopy and image processing.
(A) The extended Mediator contains three distinguishable regions; head (h), middle (m),
and tail (t). The globular density embraced by Mediator is identified as RNA polymerase II. The outline of a
projection of the previously determined polymerase three-dimensional structure is superimposed (dark line),
with the point of attachment of the C-terminal domain (dark circle) and the location of the DNA-binding channel
(c) indicated. (B) Tentative subunit organization for the holoenzyme. The model is based on available structural
Information and reported physical interactions. The surface of each subunit has been calculated by assuming
a globular shape and drawn in scale. Subunits in red have reported homologs in Saccharomyces pombe and,
with the exception of Rox3 and Srb6, also in mammalian Mediator. The yellow subunits are specific for
Saccharomyces cerevisiae.
By Bjorklund & Gustafsson, (2004) Advance in Protein Chem., 67, 43-65
Mediator and its interaction with the basal transcription machinery. The structure of the RNAPII–Mediator
complex has revealed the way in which RNAPII interacts with the Mediator complex. As shown, upstream promoter
DNA, IIB and TBP are all expected to be located at the interface between polymerase and Mediator. This implies that
RNAPII and Mediator cannot arrive at a promoter as a pre-formed complex, but must be recruited independently.
Asturias FJ. 2004. Curr Opin Struct Biol. 14(2):121-9. Review.
Enhancer-promoter communication
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
Activation of the HNF-4 Gene during CaCo-2 Cell Differentiation and Mapping of the Upstream Regulatory Region. (A)
Total RNAs prepared from CaCo-2 cells at the indicated hours after reaching confluence were analyzed by RT-PCR
using specific primers HNF-4, Enh-3’, Int.1, and ARP PO as control. Quantitation of HNF-4 mRNA levels were
performed by phosphoimage analysis and verified by real-time PCR. Values at the bottom represent normalized
HNF-4 reaction products obtained by real-time PCR from the same cDNA samples.( B) DNase-I hypersensitive analysis.
Nuclei from the indicated time post-confluent CaCo-2 cells were digested with 0 to 20 units of DNase-I, and genomic
DNA was prepared and digested with either HindIII or EcoRI. Digestion products obtained with 10 units of DNase-I
from each time point were separated on 1% agarose gels and subjected to Southern blot hybridization with the
indicated probes. The scheme below shows the positions of the major hypersensitive sites relative to the transcription
start site.
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
Nucleosome Structure Analysis of the HNF-4 Regulatory Regions in Differentiating CaCo-2 Cells. (A and B)
Nuclei from the indicated times postconfluent CaCo-2 cells and A2780 cells were digested with 0 to 170 units
of micrococcal nuclease. Total DNA was prepared and digested either with AccI (A) or MscI (B). Digestion
products obtained with 50 units of micrococcal nuclease were separated on 1.5% agarose gels, stained with
ethidium bromide, and, after photography (shown in the panel EtBr), blotted to nitrocellulose filters and
hybridized with the indicated probes (left panels). (C) Schematic presentation of the HNF-4 proximal promoter
and enhancer relative to nucleosome positions. (D) Aliquots of nuclei preparations of (A) were digested with
50 units of BglII, and genomic DNA was prepared, which was fully digested with AccI and analyzed in
Southern blots with Probe 1.
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
Order of Recruitment of Transcription Factors at the HNF-4 Enhancer and Promoter in Differentiating CaCo-2
Cells. (A) Schematic presentation of the position of PCR primers used in the chromatin immunoprecipitation analysis.
Numbers indicate the 5’ nucleotide positions of the primers relative to the transcription start site. (B) Chromatin
immunoprecipitation (ChIP) assays. Soluble chromatin from crosslinked cells was immunoprecipitated with the
indicated antibodies, and the DNAs in the immunoprecipitates were amplified (19–25 cycles) with the indicated
oligonucleotides. Autoradiographic images of the products separated on 5% polyacrylamide gels are shown.
Order of Recruitment of General Transcription Factors and RNA Pol-II to the HNF-4 Promoter in Differentiating
CaCo-2 Cells. Chromatin immunoprecipitation assays were performed with the indicated antibodies. Note that the
antibody labeled RNA pol-II (Santa Cruz Biotechnologies, sc-9001) was raised against the N terminus of the protein
and recognizes both unphosphorylated and hyperphosphorylated forms of the molecule, while CTD-Ser5P and
CTD-Ser2P (Covance H14 and H5) specifically recognize the carboxy-terminal domain of pol-II phosphorylated at
Ser5 and Ser2, respectively.
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
Stable Enhancer-Promoter Complex Formation in 80 Hr Postconfluent Cells. Complexes
immunoprecipitated with the indicated first antibodies were eluted from the protein-G-Sepharose
beads and, after dilution, were reimmunoprecipitated with the indicated second antibodies.
PCR reactions were performed with primer sets amplifying the HNF-4a enhancer (Enh),proximal
promoter (Prom), and coding region (Cod).
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
Histone Modifications and Recruitment of Acetyltransferases and the Brg-1 Chromatin
Remodeling Factor to the HNF-4 Regulatory Regions in Differentiating CaCo-2 Cells. Chromatin
immunoprecipitation experiments were performed with antibodies raised against
histone tail peptides bearing the indicated modifications (A) or antibodies recognizing
CBP, P/CAF, and Brg-1 (B).
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.
1. Poised or committed state of the HNF-4 gene
2. Recruitment of CBP, P/CAF, and Brg-1 to the
enhancer region and assembly of the RNA pol-II
holoenzyme at the proximal promoter region.
3. Unidirectional movement of the DNA-protein
complex formed on the HNF-4 enhancer along
the intervening sequences and spreading of
histone hyperacetylation.
4. Formation of a stable enhancer-promoter
complex, hyperacetylation of nucleosomes
located at the promoter, remodeling of the
nucleosome located at the transcription
start site, and release of RNA pol-II from the
promoter.
Model Depicting the Sequential Steps Involved in the Formation of an Active Pre-initiation
Complex on the HNF-4 Regulatory Region.
Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.