MCB 201 Gene Expression - Spring Semester 2003
Lecture 17 (Regulation of Transcription Initiation cont.)
Section 10.6 (RNA polymerase II (Pol II) transcription-initiation complex)
1. Figure 10-50: What are general transcription factors? Initiation factors which position Pol II at transcription initiation sites for most if not all genes transcribed by this polymerase. These factors can be separated from the polymerase itself during purification and are different from the subunits of Pol II.
A. What is a transcription-initiation complex? RNA polymerase and general transcription factors bound to the promoter sequence of DNA.
B. Many of the eukaryotic general transcription factors have been purified and characterized. The largest is TFIID, which consists of one TATA box-binding protein (TBP) and elevenTBP-associated factors (TAFs). It is not shown in this diagram, but would sit behind TFIIH in the initiation complex.
C. Stepwise assembly of a transcription-initiation complex from isolated RNA polymerase II (Pol II) and general transcription factors. It is important to note that figures like this one are primarily based on data from in vitro experiments in which purified transcription factors are added back to naked DNA. The mechanisms in intact cells are more complex. In the in vitro experiment shown here, TBP is added to the DNA to bind to the TATA region of the promoter. This marks the spot where other transcription factors will now bind. In vivo, TBP is actually part of a larger multiprotein complex called TFIID, which is difficult to purify. Note that separation of the DNA strands at the start site to create the so-called "open-complex" requires ATP hydrolysis. As transcription initiates, the carboxy-terminal domain of RNA Polymerase II (CTD) becomes heavily phosphorylated and the general transcription factors dissociate from the TBP-promoter complex.
2. Figure 10-51: TBP binds to DNA in the minor groove and it bends the DNA. Many DNA binding proteins are dimers with two-fold axes of symmetry. TBP has an interesting structure in which the monomer has a two-fold symmetry axis, and it sits on the DNA like a saddle on a horse.
3. Figure 10-50: We can now go back to this sequential diagram and see that the next factor that can bind is TFIIB, which contacts both the DNA and bound TBP, as shown in the next figure.
4. Figure 10-52: Initiation in vivo also requires TFIIA which binds on the opposite side of TBP-DNA and is thought to bind before TFIIB.
5. Figure 10-50: Next, a preformed complex of PolII and TFIIF binds to form a preinitiation complex, in which PolII is positioned over the start site for transcription. A structural model of this complex is shown in the next slide.
6. Figure 10-53: This figure is good for appreciating the relative sizes of the various components. Two views rotated 180 degrees are shown. Note that a channel is formed through which the DNA 'flows' in a sense, since the transcription complex must move relative to the DNA. The two 90 degree bends of the DNA are clearly visible in the lower image. Also, the region of DNA which can be crosslinked to TFIIF is marked. The two finger-like structures at the top of model form the active site of PolII and therefore they also mark the transcription start site. In order to create this model, data were combined from electron microscopy, x-ray crystallography and protein-DNA cross-linking studies.
7. Figure 10-50: Two more general transcription factors then bind, TFIIE, which creates a docking site for TFIIH, the next factor, which contains two helicase active sites. This activity uses the energy of ATP hydrolysis to unwind the DNA helix and to expose the template strand. This is called the "open-complex". If ribonucleoside triphosphates are added to the in vitro reaction, the polymerase will begin transcription and separate from the initiation complex. Simultaneously, another subunit of TFIIH, which has kinase activity, phosphorylates the serine and threonine rich C-terminal segment of PolII called the carboxylterminal domain or CTD. This large increase in negative charge due to phosphate addition may help to dissociate the general transcription factors and release PolII.
8. In vivo, the thinking is that a large complex about the size of a ribosome forms, and then binds to promoter DNA in a single step. This is called a holoenzyme with a size of about 2 million daltons.
8.5 The molecular structure of yeast RNA polymerase II has been solved by X-ray crystallography and a news item appeared in Science magazine, vol. 292, 20 April 2001, page 411, announcing this accomplishment. The Pol II structure was determined without (Fig 8.5a) and with (Fig 8.5b) bound DNA and nascent RNA chains. Ten subunits combine to form yeast Pol II.
Fig 8.5a: Notice the open cleft which looks and in fact functions like an open clamp. This is the active site where DNA binds and RNA is produced.
Fig 8.5b: Here the clamp has locked onto a piece of double stranded DNA and the molecular clamp has closed. Ribonucleoside triphosphates ente the polymerase complex through a pore and tunnel to the active site where they are polymerized into complementary RNA. Human Pol II has a similar amino acid sequence to yeast Pol II raise expectations that its molecular structure, at least in the core active site area of the molecule, will have a very similar structure.
Section 10.7 (Molecular mechanisms of eukaryotic transcriptional control)
9. 'Unlocking the Gates to Gene Expression' (Fry and Peterson, Science 295:1847(2002). The big question in transcriptional activation of genes buried in eukaryotic chromatin is How can the transcriptional machinery gain access to the genes hidden among the nucleosomes? The answer lies in understanding the action of chromatin-remodeling enzymes. There are two classes:
a. Enzymes that covalently modify nucleosomal histone proteins by acetylation, phosphorylation or methylation primarily, e.g. histone acetyl transferases (HATs).
b. Enzymes that alter chromatin structure using the energy released by ATP hydrolysis, e.g. SWI/SNF.
In this review article, the authors describe three different genes that are activated in different ways (see figure, panel A, B and C). The activation mechanisms differ in when the transcriptional activator recruits the remodeling enzymes relative to the time when the pre-initiation complex is formed (PIC). That may be before (panel A), during (panel B), or after (panel C) assembly of PIC, depending on the gene.
Panel A: Activation can be broken down into several steps:
1) For the yeast HO gene, the activator Swi5p remains associated with the chromatin during mitosis and in the last stage, telophase, when chromatin decondenses, it recruits the SWI/SNF chromatin-remodeling complex to the upstream regulatory region of the HO gene. SWI/SNF activity is required to recruit the HAT complex Gcn5p, also at the end of mitosis.
2) The HO gene is actually expressed during G1 phase of the next cell cycle. Histone acetylation by HAT, i.e. chromatin remodeling, prepares the chromatin for binding of second gene-specific activator SBF.
3) RNA Pol II and other general transcription factors are recruited, PIC assembly is completed, and HO gene transcription is initiated.
10. Figure 9-30b: A molecular model showing a side view of a nucleosome with DNA shown in white. The histone names mark the positions of the N-termini of histone proteins. These are sites of modification of histones by acetylation, methylation, phosphorylation and addition of ubiquitin. This region is rich in lysine residues that are frequently the target of modifying enzymes.
11. Figure 3-2b (p.52, Lodish) shows the structures of the hydrophilic members of the 20 naturally occurring amino acids. Pick out lysine. Note that it is positively charged, which should help to neutralize negative charge on phosphodiester bonds of DNA, allowing tighter folding or packing of chromatin.
12. Figure 10-58: Of the various modifications of histones, we know the most about the effects of acetylation/deacetylation. Here is a model of how this modification may play a role in transcriptional control in yeast. The key regulator is the repressor Ume6, which binds to a control element in the DNA (URS1) near a TATA element (promoter). Note the Ume6 has two domains, a DNA-binding domain and a repression domain linked by a flexible region of polypeptide chain. Its repression domain binds a multiprotein complex that contains a histone deacetylase activity (subunit Rpd3). Deacetylation of the N-termini of histones of nucleosomes in this region of DNA inhibits binding of general transcription factors to the TATA box, thus repressing transcription. Panel B: a model for activation is shown. Here the activator is a protein called Gcn4, which has a DNA binding domain that interacts with UAS and an activation domain that interacts with a multiprotein complex. Included in this complex is a histone acetylase activity (subunit Gcn5) which hyperacetylates the N-termini of histones nearby. This allows unfolding of the chromatin and gives general transcription factors access to the DNA.
