Fundamentals 1: 11:00 - 12:00 Scribe: Kosha Shah/Susan Whitt

Fundamentals 1: 11:00 - 12:00 Scribe: Kosha Shah/Susan Whitt

Monday, September 14, 2009 Proof: Myra Dennis

Dr. Ryan Gene Expression: Eukaryotic Gene Transcription Cont Page 1 of 7

RNA polymerase 1=RNAP1 or pol 1 , RNA polymerase 2=RNAP 2 or pol2, RNA polymerase 3=RNAP3 or pol 3General Transcription Factors=GTFs

I.  Introduction [S1]:

II.  Eukaryotic RNA Polymerases [S2]

a.  We have gone from having 1 RNA polymerase to 3 RNA polymerases. They are all DNA dependent RNA polymerases.

b.  They are labeled RNA Polymerase 1, RNA Polymerase 2, and RNA Polymerase 3.

c.  All three of these are large, multimeric proteins, similar to E.coli 1.

d.  They have 2 large subunits with sequences similar to Beta and Beta prime subunits of E.coli polymerase.

e.  This suggests that the catalytic site may be conserved.

f.  They all interact with General Transcription Factors, or GTFs.

g.  They all have a varying degree of sensitivity to alpha amanitin. RNA polymerase 2 is the most sensitive to this drug.

III.  Polymerase II Inhibitor alpha-Amanitin [S3]

a.  Alpha amantin comes from a fungus called the Destroying Angel, Amanita phalloides, so you don’t want to eat these mushrooms. They will block all your RNAP 2 functions in your cell.

b.  This is the structure of it. It is a bicyclic octapeptide, and it blocks elongation of RNAP 2.

c.  The three polymerases found in eukaryotes have a varying degree of sensitivity. Pol 2 is most, pol 3 is next, and pol 1 is not very sensitive.

d.  If you want to test to see if your gene is transcribed by 1, 2 or 3, this is a good way to do it. Throw alpha amantin in your culture and if it is pol 2, it should be knocked out. If it is pol 1, it is not going to be affected much. Pol 3 is similar

IV.  Yeast RNA Polymerase II Subunits [S4]

a.  This is a table from your book showing 12 subunits found in yeast RNAP 2.

b.  Each of subunits is called RPB followed by a designation 1 through 12.

c.  RPB is RNA polymerase B, which is RNA polymerase 2. If it said RPA, that would be RNAP 1.

d.  These are arranged almost in size from largest to smallest. The two large ones are listed first. They are homologous to the E.coli RNAP beta prime and beta subunits.

e.  RBP 3 has homology to the alpha subunit.

f.  RBP 4, which is involved in promoter recognition, is similar to sigma sequences.

g.  In the largest subunit, there is a repeating heptapeptide sequence on the C terminal domain. CTD stands for C terminal domain. That will be very important for the initiation elongation of transcription later.

h.  Some of these subunits are shared between all three polymerase. 5, 6, 8, 10, and 12 - these are subunits of all three polymerases. There are also individual subunits that are specific for pol 1, 2, and 3.

V.  Transcription Factors [S5]

a.  All of these polymerases interact with their promoters via protein-protein and protein-DNA interactions, again transcription factors.

b.  He read rest of slide.

c.  Like E.coli, we will need transcription factors to help find the promoter to initiate transcription.

d.  When you see TF, it means transcription factor. 3 means RNAP 3 transcription factor.

e.  These transcription factor binding sites aren’t always upstream. Some of them can be within the gene itself.

VI.  Helix Turn-Helix Motif [S6]

a.  To describe some of the types of transcription factors, the first one I want to talk about are these helix-turn-helix motifs.

b.  On the list here, here is the Trp repressor, here is the CAP protein, here is the lac repressor. This is a very common motif, especially in prokaryotes.

c.  They usually act again as homodimers and sometimes heterodimers.

d.  They have the helix-turn-helix: the two helices shown in orange or brown are alpha helices. One of them sits down in the major groove of DNA, and the other helix holds it in there. If it is binding as a homodimer, you have this DNA binding sequence.

e.  Student question: Can you explain alpha helices one more time?

f.  Answer: Alpha helices is a secondary structure of protein. There are 2 of these in this protein. One of them will sit right in to the major groove of DNA, and then you have turn, and the other sits on top of it and holds it in place. You have a tight binding when it binds its specific sequence.

VII.  Zinc-Finger Motif: C2H2 Class [S7]

a.  The second class is Zinc-Finger Motif.

b.  There are thousands of these in our cells.

c.  They have structure that has 4 amino acids, in this case two cysteines and two histidines that coordinate a zinc molecule. This DNA that is looped in between is the finger. The finger can bind to DNA

d.  They can have multiple fingers. You can have a zinc finger transcription factor with 4 zinc fingers. Each one of these fingers is going to recognize 5 base pairs of DNA: 3 that are specific for it, and one base before and one base after, so the fingers overlap with the bases on the end. If you have 4 fingers, there are 12 base pairs of DNA specific sequence that this transcription factor is going to bind and recognize.

e.  It is a very common motif.

f.  There is a variable region in which the amino acids will change. The types of amino acids in this region will dictate which bases they will recognize in the DNA. There are actually companies that will design any finger that will bind to DNA sequence you want. But they say they don’t always work, and you have to test individually, but it’s a very powerful way of potentially activating or repressing transcription from a specific gene if you can design a factor that will bind a specific sequence.

g.  Our body is full of thousands of zinc finger transcription factors. All of them recognize different DNA sequences.

VIII. Basic Region-Leucine Zipper Motif[S8]

a.  Also called BZip for short.

b.  They are mostly alpha helical in structure. In the amino terminal, there is a basic region that binds DNA in the major groove.

c.  The zinc fingers also bind in the major groove.

d.  On the C terminal end, there is the leucine part of this. These are amphipathic helices. If you have an alpha-helix, about every 7 AA, you end up on one side of this cylinder. Every 7 of these are leucine. So you have this hydrophobic face to this amphipathic structure. There is a dimer coming here where the leucines line up and you get this hydrophobic domain, and they come together and hold on.

e.  Amino terminal of binding region can fit in the major groove of DNA.

f.  You can also form heterodimers with different parts. Can have one that binds to one sequence of DNA and another that binds another, but their leucine zippers will zip them up. So you can extend the range of different sequences that this protein can recognize and activate.

IX.  bZIP Transcription Factor [S9]

a.  Here is a ribbon diagram showing these proteins scissoring going in the major groove of DNA behind the DNA helix. They are sitting on top with leucine zippers that have them attached. This is a heterodimer with the two colors representing two different proteins. It wouldn’t have to have diad symmetry because there are two different proteins with two different DNA binding regions.

b.  If you had a transcription factor binding sequence with diad symmetry, you would get a homodimer

X.  General Transcription Factors (GTFs) [S10]

a.  He read the slide.

b.  These GTFs (scores of proteins) come together to help polymerase find its transcription start site.

c.  TF represents transcription factor, II for RNA pol 2, and they are given letter designations, A, B, D, F, and H. These proteins have been isolated and they are known for RNAP 2 transcripts using a TATA box.

XI.  General Transcription Factors [S11]

a.  TFIID is the largest of these and consists of a TATA box binding protein, or TBP. It also has 8-10 TATA binding proteins, or TAFs for RNA polymerase 2.

b.  He read the slide.

c.  TBP is still used even if the promoters do not have a TATA box.

d.  TAFs help recognize a specific promoter sequence.

e.  You don’t want your promoter region genes to be bound and form higher order structure. By having these GTF around, you help the promoter stay open.

XII.  TBP is used by all 3 RNA polymerases [S12]

a.  Read the slide.

XIII. Yeast TATA Binding Protein TBP [S13]

a.  Here is a ribbon diagram showing the TATA binding protein binding to DNA. You will notice that apart from the previous transcription factor classes we talked about, TBP binds in the minor groove. When it binds there, it bends the DNA about 120 degrees.

b.  This is an ATP ripped sequence, so it helps melt and open this sequence upon binding (Not sure exactly what he said). Some people say it looks like a saddle and you are saddling up the DNA.

XIV.  Promoters for RNAP 1 [S14]

a.  Will talk about RNAP 1 and RNAP 3 and then get back to RNAP2.

b.  He read the slide.

c.  We have lots of ribosomes in our cells. Ribosomes are a mixture of protein and ribosomal RNA molecules.

XV. Transcription of rRNA genes by RNA polymerase 1 [S15]

a.  Here is a schematic of what RNA polymerase looks like.

b.  The little red region is a space “this is one gene here and another gene here. You will have a hundred, two hundred copies of these. The promoter will be right in this region”

c.  You will transcribe this large pre-ribosomal 45S pre-ribosomal RNA message. This thing gets processed to produce three ribosomal RNAs: 18S, 5.8S, and 28S. !8S will then associate with 30 proteins forming the small ribosomal subunit. The 5.8S and 28S, the 5S ribosomal RNA, which is a pol 3 transcribed gene, and 50 proteins come together to form a 60S ribosomal subunit

XVI.  Promoters for RNAP III [S16]

a.  The small 5S ribosomal RNA is a pol 3 gene. All the transfer RNAs are pol 3 genes

b.  He read the slide.

XVII.  PIC Assembly for RNA Pol III Genes [S17]

a.  Here is an example of class 3 genes transcribed by pol 3. Here is the tRNA gene and here is the 5S RNA gene. They have two sequences called Box A and Box B or Box A and Box C that are totally within the genes.

b.  You have these factors that load on to these boxes and interact and form the pre-initiation complex. You will notice that the TATA binding protein is used by all polymerases, and is part of the TF3B complex that comes in shown here.

c.  When all these GTFS are bound, pol 3 will come and you can start transcribing these genes.

XVIII.  RNA Polymerase II: General Transcription Factors [S18]

a.  He specifically read each line on slide. In addition he said the following;

b.  TFIID is composed of TATA binding protein, which is a single protein. It recognizes the promoter TATA, binding the minor groove, bending the DNA, opening up the helix a little bit.

c.  HAT stands for Histone acetyl transferase.

d.  TFIIB assists transcription start site sequence. Helps polymerase associate with TFIID and the actual transcription initiation site.

e.  RNA pol 2 has 12 subunits and once you have all that bound, you will recruit, TFIIE. TFIIE, which will help recruit TFIIH and help modulate TFIIH activity. TFIIE has ATPase activity. You need energy to drive transcription, so ATPase will cleave ATP and generate energy to transcribe.

f.  TFIIH has helicase activity, which will help melt DNA to get promoter initiation. It also has a kinase activity, which will phosphorylate this carboxy-terminal domain of the large Beta subunit of RNA polymerase. Only after that CDT domain gets phosphorylated will transcription elongation occur and polymerase will leave promoter.

XIX.  TBP-associated Factors (TAFs or TAFIIs) [S19]

a.  Based on what cell type you are working with, the TAFs can be different. There is lots of variation from cell to cell.

b.  He read the slide.

c.  The TFIID complex may come in a single step.

XX. Eukaryotic RNA Pol II Transcription [S20]

a.  Here is transcription start site plus 1. TFIID has the TATA box binding protein and all the TAFs associated with it.

b.  Here is the TATA Box, and TFIID sits on the TATA box.

c.  TFIID comes in and helps stabilize the interaction.

XXI.  Eukaryotic RNA Pol II Transcription [S21]

a.  Next, TFIIB will come in and bridge this distance from the TATA box to the initiation site

XXII.  Eukaryotic RNA Pol II Transcription [S22]

a.  Now you can bring in RNAP2. It has 12 subunits, It has TF2F associated with it. You already have TF2D, TF2A, and TF2B already loaded and now RNAP can come in.

b.  It will help melt the duplex so we can start transcribing.

c.  Once the polymerase and TF2F have bound, TF2E, the DNA dependent ATPase will come and bind. This is necessary for the energy to drive transcription. Once all this is in place, TF2H can phosphorylate the tail. Once the tail has been phosphorylated, transcription can proceed, and these factors are left behind. TF2H is a 9 subunit GTF. It has helicase activity to help unwind DNA and also has protein kinase activity to phosphorylate the carboxyl terminal domain tail of RNAP2.