The Eukaryotic Gene Transcription Machinery:Mechanism and Regulation

Zhen-Qiang Pan, Ph.D

March 24, 2005

1) An overview of the eukaryotic gene transcription

Outlines:

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 provideexperimental 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 at2.8 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 representationof 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 microscopestructures (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

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.

Tail

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

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-PCRusing 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 theindicated probes. The scheme below shows the positions of the major hypersensitive sites relative to the transcriptionstart 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 promoterand 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.

Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.

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. Complexesimmunoprecipitated with the indicated first antibodies were eluted from the protein-G-Sepharosebeads 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. Chromatinimmunoprecipitation 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.

Model Depicting the Sequential Steps Involved in the Formation of an Active Pre-initiation Complex on the HNF-4 Regulatory Region.

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 thepromoter.

Hatzis P, Talianidis I. 2002. Mol Cell 10(6):1467-77.