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Lecture 4

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 14 2012 Lecture 4 Dr. Lasko Genes in a eukaryotic DNA sequence are normally in a tightly condensed chromatin structure which must first be loosened up for the transcription machinery to get inside the DNA grooves  RNA polymerase is not the only enzyme involved, getting RNA Pol to the DNA requires the work of many more protein factors o General TFs play a role in targeting and recruiting RNA Pol to the promoter o Activators help recruiting RNA Pol as well as promoting the assembly of other subunits of the complex o The Mediator complex helps mediate interactions between RNA Pol and distant enhancers (like sigma 54 promoters in prokaryotes)  Activating transcription involves getting from the beginning state on top to the open and ready state in the bottom with all proteins assembled Initially, general TFs work along with RNA Pol; this was studied through in vitro assays  TBP (TATA Box Binding Protein) is a subunit of TFIID which binds to the DNA  Other TFIIs (B, F which carries RNA Pol II, E, H) associate with the complex now attached to the DNA o Remember, the CTD is responsible for allowing elongation to occur  The pre-initiation complex is the material around the DNA (i.e. Pol II and all of the general TFs) Transcription of a gene is regulated by regulatory sequences which can be quite far from the promoter:  These can be grouped into different types: o Promoter proximal elements are the closest to the promoters o Enhancers (which activate transcription) and silencers (bind repressors which turn off transcription) tend to be farther away from the promoter (as much as 50 kb) but can also be 1 th BIOL 300 September 14 2012 Lecture 4 Dr. Lasko located within introns or downstream of the transcription termination site Pol II core promoter elements include the following:  The TATA box is located 31-26 nucleotides upstream of the transcriptional start site; it was named because it contains a TATA sequence o Not all eukaryotic promoters have a TATA box (in fact only a minority have one, but they still represent a substantial amount)  There are other elements which can help catalyze the formation of the pre-initiation complex o The initiator element, Inr, overlaps with the start site, but doesn’t have a strong consensus sequence  In mammals, we can see that it only has to be two pyrimidines, following by A, then anything, then T or A, then two more pyrimidines  They don’t work by themselves, they work in association with other promoter elements o The downstream core promoter elements again don’t have a strong consensus site  The BRE, or TFIIB Recognition Element, is a GC rich sequence which is an example of a core promoter element  Promoters typically have some subset of these 4 potential elements o If they don’t have a TATA Box, they have some of the other three, while if they do have a TATA box, they don’t have to have any others o These components associate with different subunits of the pre-initiation complex (mostly through TFIID) The TATA Box has a strong consensus sequence, but again the site is not the same 100% of the time  The only nucleotide that is always there is the second T; if it is missing, TFIID will not bind  The other nucleotides are not always there; TFIID can still bind even with a mismatch at one of the highly conserved regions o The more the mismatches, the weaker the promoter element (there are probably very few elements missing both the 1 and 2 which are both over 90% frequent)  The transcriptional start site is 25 or so nucleotides after the end of the TATA box 2 th BIOL 300 September 14 2012 Lecture 4 Dr. Lasko o If you artificially removing the start site, transcription will still take place, but at a different site which is still 25 base pairs downstream of the end of the TATA Box  This is because RNA Pol’s active site happens to be 25 nucleotides downstream of where it binds to the TATA Box  The TATA Box was the first discovered core element in 1979; it was found to bind TBP, a subunit of TFIID  TBP can bind variety of AT-rich sequences. Sequence TATAAA (+/- I mismatch) found in 43% of Drosophila promoters o The TATA Box does in fact resemble a lot of prokaryotic promoters The initiator element (Inr) does not have a strong consensus (stronger in drosophila than in mammals)  Overlaps the start site  Pyrimidine rich  Occurs in 69% of Drosophila promoters  Can function in the absence of the TATA box  Can synergize with the TATA box when 25 base pairs apart o When >30 base pairs away, no synergy can occur  Binds the TAFI and TAFII subunits of TFIID  When TFIID binds TFIIA, this enhances TFIID binding to Inr The downstream core promoter element (DPE) usually works in conjunction with an Inr  Spacing between DPE and Inr is critical once again; a single base deletion or insertion decreases the rate of transcription o This is because proteins are of a fixed size; the binding site of TFIID for the Inr and its binding site of DPE are located approximately 25 base pairs apart from one another o If the distance between the elements get changed, the protein will have to strain to fit onto them properly, thus lowering the affinity and lowering the rate of transcription  Also, because of the helix shape of DNA, a deletion or insertion will result more or less turns of the helix, meaning not only is the length changed, but the angle as well, making it even harder for the protein to bind 3 th BIOL 300 September 14 2012 Lecture 4 Dr. Lasko o DPE binds to the TAF6 and TAF9 subunits of TFIID and is present in about 40% of drosophila promoters (less in mammals) The BRE is a 6 or 7 base pair long GC rich region which does not interact with TFIID, it interacts with TFIIB (which in turn binds TFIID)  TFIIB interacts with the major groove in the DNA helix  Can mediate activation or repression  Discovered recently, in 1998, through analysis of the crystal structure of TBP- TATA-TFIIB complex Remember, the number of these elements in a promoter, as well as how well they match the subunit, will determine the strength of the promoter  The strength of the promoter, in turn, will determine the rate of transcription (the likelihood of attracting RNA Pol II)  Two of these regulatory elements (Inr and DPE) actually either overlap the start site or are inside the gene which is being transcribed (i.e. will be present in the RNA) o Housekeeping genes tend not to have all of these elements The promoter proximal elements (PPEs) are the closest regulatory sequences to the promoter:  This is the PPE region in a higher eukaryote (TATA Box and other associated regions are not too far away)  Many higher eukaryotes have PPEs 100 to 200 base pairs away upstream of the transcription start site o E.g. the CG rich box and the CCAAT box are shown here  They tend to bind broad-range activators which are NOT gene specific o They have binding sites for either Sp1 or C-EBP/NF-Y which are not general TFs, but increase the level of expression  Many promoters have PPEs with binding sites for these activators The first example of an enhancer was found in the sequence of a virus which infects humans (simian virus 40, i.e. SV40)  Enhancers are
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