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Lecture 11

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Lecture 11 -Gene regulation II: Transcription Factors and MicroArray Technology SPs: Figs 12-34, 36, 37, 38, 39 Vocabulary: transcription factor types/DNA binding and activation domains/hetero vs. homodimer/ combinatorial control/microarray technology Today will talk about transcription factors and something used in genomics called MicroArray technology. MicroArray technology is a high input genomics technique that can be used to examine the expression of every gene in an organism at the same time. So if an organism has 25,000 genes, using MicroArray technology you can find out what's happening with each of those at once. Transcription factors are proteins – they are trans acting factors that diffused through the nucleoplasm or in bacteria through the cytoplasm of the cell and they have at least two functional domains. The first has to do with their role in transcription and one of their domains is referred to as the DNA- binding domain. This is a sequence specific interaction of this particular protein with a specific DNA sequence. They have recognition sequences that are very different from one another in terms of the nucleotide sequence, but the structure of the sequence is typically present as a palindromic DNA sequence. A palindromic sequence is something that has access to a twofold symmetry. If you look at the double-stranded DNA, the sequence reads 5’ AGAACA 3’ and what you can see on the opposite strand of the same polarity 5’ AGAACA 3’ so it is the same sequence that is repeated in a converging order. This is what most transcription factors bind to; not any specific nucleotide sequence but one that is present in a palindromic structure. The “nnn” in the middle represents that the palindromic structure is sometimes preserved and sometimes it is not and the sequence can vary from 3 to 15 nucleotides long so quite a lot of information can be derived from such a sequence. Transcription factors also form dimers i.e. two molecules get together - they can be the same molecule in which case they are called homodimers or they can be two different transcription factors that get together and form a complex called a heterodimer. The second facet that every transcription factor has is something called an activation domain. This can be a little misleading sometimes because this is the domain that interacts with one or more proteins to promote transcription (to activate transcription) but some transcription factors actually served to turn off a gene or repress it. It is still an activation domain but what it does is repressed. Figure 10 – 10 Where do these sequences occur in double-stranded DNA? In figure 10-10, you can see a schematic of what the double helix looks like. It is not very symmetrical structure. It has a wide major groove on one side and on the other side has a minor groove. You can see that the major groove has an open structure that allows the nitrogenous bases that exist there to be easily accessed for example by transcription factors. Figure 12 – 18 from Brooker textbook At the top, the figure shows a regular double helical structure of DNA with the major groove on the top and minor groove on the bottom. In the side or cutaway view, you can see that the major groove is wide and the minor groove is narrow and within the major groove, the nitrogenous bases can be easily accessed and they look like this. What you can see is that there are a few circles around substituent groups such as hydrogen bond donors, hydrogen bond acceptor, hydrogen atoms, and around a Methyl group. There is information that is imparted by the nucleotide sequence. A key point is that the sequence specificity for a protein that is a transcription factor resides in the type and order of the bases that are present in DNA. The game mastermind illustrates this idea that many combinations from only a few colors or a few nitrogenous bases can produce quite a number of different specific binding sites. On a DNA molecule that same idea might look something like Figure 12 – 18 in the broker textbook. It shows the double helix and part of the protein structure incorporated in the DNA double helix and it shows some of the sidechains of the amino acids that are closely juxtaposed to some of the nitrogenous bases. The code for DNA that we are using is -Green is a hydrogen bond acceptor, -Orange is a hydrogen bond donor, -Red is a hydrogen atom, and -Blue is a methyl group. For many types of DNA-binding proteins such as restriction enzymes (which are ways to cleave DNA in a reproducible fashion), there's a restriction enzyme that recognizes the sequence ACGT so the color code for that looks something like the first image. Here, there are certain functional groups that are presented within the major groove, and that particular restriction enzyme can come in and recognize the sequence and partake in binding and cleavage. An example for seed specific transcription factor (in plants) recognizes the specific sequence called “CAT G CAT” (not on test). This is another example that shows different color combinations that allow the transcription factors to bind specifically. The next image shows a Drosophila developmental biology transcription factor and it's pattern is around 8 or 10 nucleotides and is very different because different groups are present depending on the nucleotide sequence. These are things which determine the specificity of binding of the transcription factor with its DNA sequence. There are 3 main classes of transcription factors: 1. Zinc finger transcription factor The first major class is called zinc finger transcription factor. Figure 12 – 37 shows a ribbon model of this TF. Zinc is a metal ion. Parts of the molecule contain 2 of the particular amino acid cysteine and 2 histidine molecules in a part of the protein chain. When Zinc is available, imagine the following: your arms spread wide open represent one long protein molecule. At the elbows of one arm are the 2 cysteine molecules and at the other elbows are 2 histidines which are very far away at the same protein molecule. A molecule of zinc is available somewhere around the middle of your body. What happens is that as the coordination of the zinc ion with the cysteines and the histidines takes place, you get the zinc molecule coordinating the binding at the elbow position and when it is done that, it projects the finger or loop that goes up and projects into the major groove of DNA. The bottom image shows subsequent major grooves of DNA where a particular transcription factor has this type of zinc finger structure. You can see parts of the molecule that are in place in the major groove and not only are they binding at one place but binding along subsequent major grooves in the same molecule increases the binding specificity. That's how the zinc finger works. There are many zinc finger transcription factors that are conserved across all higher eukaryotes and do a variety of different things. 2. Helix-Loop-Helix Transcription Factor The second major group is referred to as helix loop helix transcription factor. What you see in figure 12 – 38 are two of these molecules. The basic region confers binding specificity. There is a helix loop (or turn) and then another helix and so, seen in this figure, there are two that are present along the same region of DNA so they bind as dimers. The basic domain interacts with DNA to determine sequence specificity. They're often present as heterodimer i.e. as different molecule from different families that get together and promote binding. Because different molecules can get together and interact with one another, this is a very useful thing for the cell to do because it provides the opportunity for something called combinatorial control (combinations). Figure 12 – 39 – Let’s assume that you have one transcription factor from family A. Two molecules of the A-A homodimer get together and have a certain binding specificity. Likewise, suppose you have another gene encoding molecule B and two B molecules can get together to form a B-B homodimer with a different binding specificity. We are able to form heterodimers i.e. a molecule of A and a molecule of B can get together to form A-B heterodimer and now you've extended the range of your ability to detect different sequences. With only two proteins, you can bind 3 sites so obviously with more members of the gene family, the number of possibilities you have goes up quite dramatically. This is a way to affect the function of many different genes by using different combinations of your transcription factors and this is known as combinatorial control. 3. The Leucine Zipper Motif The third main class of transcription factors is known as the Leucine zipper motif. Leucine is an amino acid and it is a hydrophobic side chain. In a leucine zipper motif what you find is that the leucine is not found randomly but rather at every 7th amino acid for about 40 amino acids or so in a row. You may have six or seven or so leucines that are strategically positioned 7 residues apart. The alpha helix positions these leucines on the same side of the molecule. Looking at the figures, you see that around the helix there is about one turn of the helix in a protein in the alpha helix about every 3 1/2 residues. So if you go from A to B to C to D and come back to the position that you started at, it comes back to the same region. You cannot have half a residue of course, but obviously a multiple of 3 1/2 is 7 so if you have a leucine at every 7th amino acid, this positions it such that if the first one was position A, then 7 more is going to position it right under the A, etc. So if you look at it in cross-section, all the A's are on the same side of the molecule. Likewise, if you have another molecule where the leucine is present at D, all of those leucines are going to be in the D position. Now, the leucine zipper motif is a way for two proteins to get together and do something quite natural based on their physiochemical properties; they zip up. This is why it's called the leucine zipper. With the leucines being hydrophobic, the interaction of these two molecules with each other is such that they are shielding themselves from the water in the aqueous environment. The two molecules with the leucine zipper motifs want to get that region away from water and do so by interacting with each other to make homodimers or by interacting with another type of leucine zipper to form heterodimers. This motif is used in something called a bZIP family. The 'b' stands for basic domain and ZIP stands for leucine zipper motifs. There's a coiled coil region formed by the leucine zipper and it is important for dimerization. The b domain (basic domain) contains basic amino acids like lysine and arginine and it mediates the specificity of DNA-binding. This is how the bZIP class of factors utilizes the leucine zipper motif to make dimers. Now let’s talk about activating gene expression versus repressing it. A Cis- acting sequence is a sequence of DNA to which something might bind. If the Cis acting sequence has a bound molecule present (i.e. it is bound specifically to some activating transcription factor), this prevents the binding of another type of molecule (that might be a repressor). Quite often there is a competition for binding and ultimately, whether or not a gene is expressed or how much it is expressed depends on the relative strength of the binding (e.g. does the activator bind more strongly that the repressor, how many positive factors are involved and what is the strength of their binding, or how many negative factors are involved and what is the strength of their binding, etc.) Figure 12 – 44 In eukaryotes, promoters are very busy places. There are many Cis-acting sequences. There are potentially many transcription factors or small molecules /ligands that the cell may have all the time, or only transiently, or only in response to certain things. This affords the ability of the cell to fine tune the expression of genes. Maybe it's appropriate for the cell to be going fast as fast it can and to make as many transcript as possible or maybe it is more appropriate in other circumstances for it to only be at 20% of its level and other circumstances be completely off. Figure 2 – 47 cartoon Very elegantly sets the stage to start talking about a revolution that has taken place in biology over the last 10 years or so. Years ago, the scientist back then could be equated to a fisherman. Essentially, they can only fish for one fish at a time or in a science sense, they could only study one gene at a time and devoted their entire scientific career to that one gene. About 15 or so years ago came a very instrumental technology and breakthrough which created some new types of departments in universities. These technologies are c
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