These Slides Are Numbered Based on the NEW Powerpoint Dr. Ryan Used in Class
Fundamentals I
9/18/08
Dr. Ryan
10:00-11:00
These slides are numbered based on the NEW powerpoint Dr. Ryan used in class**
There’s a lot to cover and get right to it.
Slide 2: Eukaryotic RNA polymerase
• I mentioned yesterday that in eukaryotic there are three RNA polymerases. They are called RNA polymerases. There are three DNA dependent RNA polymerases: Labeled RNA Pol I, II, and III
· Each of these their own sets of promoters and genes that they transcribe.
· All 3 are big, multimeric proteins (500-700 kD).
· All have 2 large subunits which are very similar to prokaryotic. RNA polymerase subunits b and b' which we talked about yesterday.
· And the catalytic site may be conserved between prokaryotes and eukaryotes.
· All of these polymerases interact with general transcription factors-GTFs. WE will go into detail on what they are.
· How do you discriminates between these three? How do you know if they gene they transfer by Pol I II or III?
• There is drug called a-amanitin and Pol II shows the most sensitivity. This is an inhibitor of pol II. a-amanitin is a bicyclic octapeptide and blocks the elongations.
Slide 3: Lecture Part 3 RNA polymerase II inhibitor a-amanitin
· The sensitivity is shown there.
· Pol II is more sensitive than Pol III and Pol I is hardly sensitivity at all.
· This drug a-amanitin comes from the mushroom called the “destroying angle.” So if you’re picking mushrooms for your morning omelet, stay away from these.
Slide 4: Yeast RNA polymerase II subunits
· These are the subunits of RNA Pol II from yeast. There is a total 12 subunits.
· The nomenclatures is RNA.
o This is RPB1, that’s for RNA Polymerase. B stands for the Pol II. RNA Polymerase I would be RPA 1-12. For Pol II subunits, they are all RPB and then the subunit 1-12.
· The first subunit shares homology with the beta prime subunit of prokaryotes.
· One of its important features is the C-terminal domain, its repeating heptapeptide, has lots of serines and theorinines in it that get phosphorlylated from the process of going through transcription initiation to the actual start of transcription. We’ll talk about that later.
· The second subunit and these are labeled 1-12 according to their size, they discovered or purified these later.
· The second largest subunit RPB2 shares homology with the Beta subunit of E.coli. It has the NTP binding site and forms half of the catalytic site with the RPB1.
· There are two subunits that share similarities (Dr. Ryan corrects himself by saying one shared similarity) with alpha that’s RPB3.
· The promoter recognition has homology to the sigma factor is RPB4.
You can see, number of these factors are shared between the three polymerases: RNA Pol I, II, and III.
Slide 5: Transcription Factors
· In eukaryotes, the transcription factors are going to very important. All three polymerases interact with transcription factors.
· A lot of these interactions are protein-protein interaction or protein-DNA interactions.
· We’ll talk a lot about types of transcription factors and these general transcription factors used by all these polymerases.
· These transcription factors (TFs) recognize and initiate transcription at specific promoter sequences. So when 6 billion base pairs of DNA, these transcription factors are going to help land the polymerase on the promoter to initiation transcription.
· Some transcription factors bind to specific recognition sequences within the coding region. So they are not upstream where the promoter is seen in prokaryotes. Sometimes they bind internally to the genes.
Slide 6: Helix-Turn-Helix Motif
· I would like to talk about three classes of transcription factors.
· These are figures from your book.
· They talk about these three classes.
· First is the helix-turn-helix motif. These transcription factors all bind DNA and what’s special about these is the helix-turn-helix motif is shown in orange in these figures.
· And these are two alph-helices that are separated by beta turn.
· The first helix sits down in the major groove in DNA, the second one locks in place. You get very specific type of binding. Most of these helix-turn-helix motif bind has homodimers or heterodimers.
· Yesterday, we talked about DNA having dyad symmetry, having inverted repeats.
An inaudible question is asked about not having the exact slides.
Answer: These are in the start of Day 4. This is the lecture of combo of 3rd and 4th lectures. All lectures are in one of these two lectures. They are mixed.
So just listen carefully. You will have a copy of these available to you.
Another question: paraphrase: Is there a PowerPoint with your notes that we can get?
A: It’s written in your book. You see when you get a test questions and you see helix-turn-helix or alpha helix beta turn alpha helix you will know what it refers to.
Another question: paraphrase: What is important ?
A: I won’t ask you anything that I don’t have on this slide or is not in your book in the reading material.
Back to lecture:
· Alright, by forming a homodimer or heterodimer, you can increase the specificity of DNA binding with these factors.
· One dimer binds one sequence or one protein in the homodimer can bind its cognate sequence on the other side and they have an interaction domain so you can increase the specificity of binding of these factors.
· Now we talked about these factors yesterday, some are of these are prokaryotes and eukaryotes that have these helix-turn-helix.
· Down here, is the cap protein that we talked about.
· The activator protein of the lac operon. It has the helix-turn-helix motif.
· Here is the trp represson and lac repressor that we talked about yesterday.
· Eurkarotic helix-turn-helix motif that is called the antrion (sp?).
o Helix-turn-helix motif is located in the homodomain.
o This is important for DNA binding.
o The specific sequences upstream are very important developmental genes.
Slide 7: Zinc-Finger Motif : C2H2 Class
· The second class of transcription factors is these Zinc-Finger transcription factors.
· There are 1000s of these in our genome.
· The typical structure is a pair of 15 amino acids apart separated by about 12 AA in the finger, and 2 histidine, 2 or 3 amino acids apart.
o There is a zinc atom that coordinates these 4 amino acids.
o Now the actual DNA binding part of the zinc-finger is located in the 12 AA space (between the cystiene and the histidine).
· Here’s the actual structures.
· So it’s got an alpha helical structure.
o Each of these fingers can contact 5 base pairs of DNA, and these fingers always come in 2 or more fingers together.
o So you can have up to 17 fingers in some of these transcription factors. So if you have multiple fingers they can bind/interact longer sequences of DNA.
o Now they each interact with 5 base pairs, but they have a tight interaction with three and interact with a neighboring one.
o So each finger, if you have 3 fingers are together they actually interact with specific 9 base pair sequence in the DNA.
o If you have 4 fingers, you would have 12.
o The middle finger is has interaction with the neighboring. Each contacts with three specific bases. S
o If you mix and match these fingers- there’s a company do this- they can build you a transcription factor that will bind any DNA sequence you want to make- so they say.
· So they again they use these fingers, there multiple finger they can bind to longer and longer stretches of DNA specifically.
Slide 8: Region Leucine Zipper Motif bZip
· The third class I would like to talk about is the basic region leucine zipper motif (bZIP). These transcription factors are mostly alpha helical in structure.
· They have a basic region that makes contact with DNA.
· And the similar to the helix-turn-helix motif and the zinc finger factors, these bZIP factors also bind in the major groove of DNA.
o All three of these major classes all bind to major groove of DNA.
· Now what’s special about these is that the single factor will dimerize with another bZIP factor.
o They do this via this alpha helical domain called the leucine zipper.
o If you turn this alpha helical regions and look at it, look at the amino acid that are located- every 7th amino acid is a leucine.
o Now the Leucine all lineup on one face of alpha helix. It’s called an amiphatic helix. It’s very polar on one surface. On the surface with leucine, it’s very non-polar. Non-polar on the other surface. **NOTE: this is what I got out of what he was trying to say. He had broken thoughts. ***
o See the two leucine surface come together-zip together. You can picture the zipping action. That’s why they get the name bZIP.
o So these can bind has homodimer or heterodimers. You can get two different bZIP coming together.
o Their DNA recognition sequences may be different. So when they contact the DNA, you can be specific in your DNA binding.
Slide 9: bZIP transcription factor
· The next figure is from your book showing a heterodimer coming together and interacting in the major groove of DNA.
· Here is the basic region sitting in the major groove.
· Here is one of the proteins, and there’s the second one behind the helix sitting in the major groove. This is would be the leucine region.
· They cut of the rest of the protein which would have activation regions that will activate transcription.
Again, there’s description in your book for all three of those.
Slide 10: General Transcription Factors
· Now the general transcription factors help position the RNA Polymerases on transcription initiation sites. (The next few slides are from the end of Lecture 3)
· These general transcription factors help the polymerase come in and land on the initiation site and form along with the polymerase a transcription- initiation complex. The nomenclature is determined by the polymerase that they are associated with.
o So, the TF- transcription factor, the II means it associates/ or its general transcription factor for RNA polymerase II.
o And then various complex of the proteins that have been isolated, and as they were purified and given letter designation of TFIIA TFIIB etc.
o We will in more detail in a moment.
So again these help the polymerase associate with the promoter find the initiation site.
Slide 11: General Transcription Factors
· Of the general transcription factors, TFIID is the largest and consists of a TATA box binding protein, that is abbreviated TBP.
· It has 8-10 transcription TATA box binding protein associated factors which are abbreviated TAF.
· Again the II there for TAFs II are because they are isolated from TFIID of RNA pol II.
· The TFIID is composed of TBP and the associated TAFs. Those together make up the TFIID.
• TBP is a “universal transcription factor.”
• It associates with promoters of all three RNAPs (pol I, II, III)
• It is also used even though it’s called the TATA box of binding proteins, it’s also used with promoters that don’t have a TATA box. These promoters that don’t have a TATA box, have to rely on other general transcription factors that help the polymerase find the initiation site.
• TFIID has two roles:
• Foundation for the transcriptional Pre-initiation complex.
• Prevents nucleosome stabilization in the promoter region.
• If you have to transcribe a gene, you don’t want nucleosomes to bind there and wrap it all up. So if it’s gene that’s expressed all the time, these so called “housekeeping genes,” you want to have this factor around keeping the nucleosome from closing up the promoter gene.
Slide 12: TBP is used by all 3 RNA polymerases
TBP- TATA binding protein is used by all three polymerases. It has different names depending on the other proteins its associates with.
· When it’s in a Pol I gene it’s called : SL1 factor.
· When it’s interacting with Pol III, its called Transcription factor III B.
· We have already talked about Pol II, it’s TFIID.
· It does not always to bind to TATA boxes. Pol I and Pol III, and sometimes Pol III promoters don’t have TATA boxes. But, TBP is still used as a general transcription factors.
Slide 13: Yeast TATA binding protein TBP.
· This is diagram of the yeast TATA binding protein. What’s unusual is that TBP binds in the minor groove. Previous transcription factors we talked about were all major groove binders.
· This is actually bound in the minor groove, while in binds it bends the DNA by 120 degrees. It also melts this region. If this region is usually A-T rich area, it helps melt this region upon binding. As it binds, it’s got amino acid chains that go in the minor groove and that helps pry the DNA apart at that region.