MCB 201 Gene Expression - Spring Semester 2003
Lecture 16 (Regulation of Transcription Initiation cont.)
Section 10.5 continued (Eukaryotic Transcription Activators and Repressors)
Some examples of types of transcriptional factors follow. These types are classified according to their DNA-binding motifs. DNA binding domains are classified into a variety of structural types based on the components of these domains that bind to the DNA, usually in the major groove. In this region, hydrogen bonds and van der Waals interactions occur between atoms in the protein and in the DNA. Contacts in the minor groove are also made by some transcription factors. X-ray crystallography has given us detailed information on these complexes between transcription factor and DNA.
1. Figure 10-40: Homeodomain proteins. Base pairs in DNA that contact
protein atoms are shown in white type against the red ribbon that represents
the DNA backbone, and contact residues in the protein are shown in white
segments of the blue ribbon that represents the peptide backbone in this
figure. The name homeodomain comes from a group of transcription factors
that are major determinants of development. They are encoded by genes called
homeotic genes, studied in detail in Drosophila, all of which encode the
same structural motif, called a homeodomain (region of 60 amino acids).
The homeobox sequence in the DNA encodes the homeodomain of these proteins.
The homeodomain, as shown in this figure, is a DNA-binding domain. Mutations
in homeotic genes produce dramatic changes in development, i.e. transformation
of one body part into another.
Figure 14-33: Antennapedia homeotic mutant fly in which antennae are transformed
into a pair of legs.
2. Figure 10-41: Zinc finger proteins. The name of this structural motif
comes from short lengths of polypeptide chains that fold around a central
ZnII ion (black dots), producing a compact domain. The ZnII ion is bound
by cysteine and histidine residues. These proteins usually contain multiple
Zn fingers that bind in the major groove of the DNA double helix. Note that
two different types of Zn finger proteins are shown in this figure. Part
A shows a monomeric type (C2H2, which stands for the two cysteines and two
histidines that bind the ZnII ion) and Part B shows a C4 type (four cysteines
bind the ZnII ion) which consists of two subunits which may be the same
(homodimers) or different (heterodimers). Notice that cylinders are used
to symbolize the alpha helices and ribbons with arrowheads for beta strands,
two important secondary structures of proteins. Steroid hormone receptors
are C4 zinc-finger proteins, along with more than 100 other transcription
factors.
3. Figure 10-42: Here is yet a third kind of Zn finger, called a C6 zinc-finger protein, of which Gal4 is an example. Two monomers bind the DNA to form a homodimer and the monomers interact to form a coiled coil perpendicular to the DNA helix. The coiled coil is a common stabilizing kind of interaction between two monomers that involves amphipathic alpha helices. In these kinds of polypeptide helices, hydrophobic side chains line up on one side of the helix, and to make a coiled coil, two of these amphipathic helices, one from each monomer, interact along these 'greasy strips' of hydrophobic side chains (see Figure 3-9a).
4. Figure 10-45: Part A: Heterodimeric transcription factors increase
gene-control options. The C4 zinc finger proteins are examples of proteins
that can form heterodimers. In some heterodimeric transcription factors,
both monomers have the same DNA binding domain and different activation
domains, which are then brought together into a single transcription factor
when the heterodimer forms. Things get more interesting when monomers with
different DNA binding sites come together, which increases the number of
possible DNA sequences that a family of TFs can bind. These interactions
are called combinatorial, a math term. For example, If three different monomers
interact two at a time, six different DNA binding sites will be recognized
and six combinations of activation domains will be created.
Part B: This figure also illustrates the effect of inhibitory proteins which
interact with one monomer of the heterodimer. When these inhibitory factors
are expressed in cells, they repress the transcriptional activation by TFs
to which they bind.
5. What is the activation domain of a transcription factor? This is the domain of the TF that activates transcription, often by directly binding to RNA polymerase, when the TF is bound to it's DNA control element. The Gal4 protein that we have been discussing has an activation domain rich in acidic amino acids (Asp and Glu). These domains are essentially unfolded, or unstructured, until they interact with a co-activator protein. Then the activation domain folds into an amphipathic alpha helix that contacts a complementary binding surface on the co-activator protein.
6. Figure 10-46. Here is a specific example, the CREB acidic activation domain bound to its co-activator, CBP. CREB stands for cAMP response element-binding protein, and CBP is CREB-binding protein. Before these two proteins can interact, some regulatory steps occur. When cAMP levels increase inside the cell, protein kinase A binds cAMP and becomes active, phosphorylating a specific serine residue (phosphoserine 133, pS133 in figure) in the CREB activation domain. This allows the domain to interact with CRB. When it does, two crucial alpha helices are formed in CREB (shown as pink spirals here) which wrap around the interacting domain of CBP. Note the interesting representation of the interacting surface of CBP, shown as the water accessible surface with basic R groups shaded blue and acidic R groups shaded red.
7. Figure 10-47. Here is a good example of the effect of a ligand on the conformation of a regulatory domain of a transcription factor called RXR, which belongs to the steroid hormone receptor family (C4 transcription factors). Panel A shows the more open structure of this domain without ligand. In this configuration, this domain is a repressor domain that inhibits transcription. When the ligand, retinoic acid (Vitamin A) binds, the conformation of the domain changes, particularly those parts shown in yellow. Now this domain can stimulate transcription.
8. Figure 10-48. What do we know about the complexes that form on the DNA sequences known as enhancers? When proteins bind to these sites, transcription is stimulated or enhanced. These turn out to be rather complex sites and the term enhancesome has been given to these multiprotein complexes bound to DNA. What are some of the typical features of these sites? The length of DNA involved can range from 50 to 200 base pairs and has binding sites for several transcription factors. The DNA is bent by the binding of HMGI proteins which bind in the minor groove and this binding is not nucleotide sequence-specific. These proteins are called architectural proteins since they are required to build these complexes. The bending of the DNA allows the transcription factors to interact with one another, i.e. cooperative binding is achieved. Even if individual proteins in the complex have relatively low binding affinities for the DNA , the entire enhancesome complex can become stable due to these cooperative interactions. In the particular complex shown here, two of these subunits, p50 and p65, compose a heterodimer known as NF-kB. This transcription factor upregulates genes involved in inflammatory responses.
9. Here are two examples from the E. coli lactose operon regulation, that are simpler than the eukaryotic enhancesome, but at the same time accomplish upregulation of transcription by cooperative interactions:
- Figure 10-17: Cooperative binding of E. coli RNA polymerase and cAMP-CAP
to the lac promoter. Both RNA polymerase and cAMP-CAP individually have
low affinity for DNA. However, When they interact with each other, they
form a complex that binds much tighter to the promoter region than either
by itself. This is called cooperativity. Protein-protein interactions between
cAMP-CAP and the alpha subunit of RNA polymerase cause shape changes in
both proteins that cause them to fit better on the DNA.
-Figure 10-18: cAMP-CAP binds and bends the DNA double helix through an
angle of approximately 90 degrees. The CAP complex is a homodimer, as you
may be able to see in this figure. The bending of the DNA may help the RNA
polymerase bind nearby and separate the strands of the double helix in preparation
for transcription. Note that when the amino acid residues shown in yellow
are mutated, cAMP-CAP cannot activate transcription, suggesting that these
residues may interact with the alpha subunit of RNA polymerase.