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Lecture

Lecture 5

5 Pages
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Department
Biology (Sci)
Course Code
BIOL 300
Professor
Siegfried Hekimi

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th BIOL 300 September 17 2012 Lecture 5 Dr. Lasko Stepwise assembly of Pol II transcription initiation complex in vitro:  TBP is a subunit of TFIID which is very hard to purify; it was the first known compound to bind to the TATA box of the promoter o Remember, in an intact cell, TFIID binds before TBP  TFIIA binds right after (not shown here)  Recruitment of TFIIB, which binds to the TBP but also to DNA on either side of the TATA box o Some promoters have a BRE sequence 5’ of the TATA box which binds TFIIB o TFIIB is critical for start site selection because they bind to specific promoter sequences; as the rest of the pre-initiation complex assembles, they assemble around the site established by these two proteins  Recruitment of Pol II comes in conjunction with TFIIF, which stimulates a CTD phosphatase activity thus facilitating incorporation of unphosphorylated (inactive) Pol II into the PIC (pre- initiation complex) o Recruitment of Pol II is done in an inactive form because you don’t want it to start moving just yet  Once Pol II is in the PIC, TFIIB antagonizes the stimulation of the CTD phosphatase activity of TFIIF o This will facilitate the accumulation of Ser 5 phosphorylated CTD when TFIIH joins the PIC later on  TFIIE is essential for recruitment of TFIIH, and stimulate a CTD kinase activity, helicase and ATPase function of TFIIH o The CTD kinase will activate Pol II, the helicase will unwind the DNA allowing Pol II to move, and the ATPase likely powers the helicase activity  THIIH has a DNA dependent ATPase activity, a kinase activity (for the CTD of Pol II), and 2 subunits with a helicase activity  Transition from the PIC to an elongation complex is associated with phosphorylation of the CTD (by TFIIH) and release of GTFs o Once the elongation complex is made, all general TFs will leave, except for TBP, which accomplishes more efficient re-initiation Now we must consider the transition from a gene turned off to a gene turned on  When off, the gene chromatin is highly condensed, making it hardly accessible by Pol II 1 th BIOL 300 September 17 2012 Lecture 5 Dr. Lasko  To be turned on, the gene has to have its DNA in a more open conformation which favors formation of the PIC  Transcriptional regulators will modify the accessibility of the DNA to transcription machinery o Activators promoter this association while repressors inhibit this association  The blue and green blobs on this diagram are regulators o Activators have a DNA binding motif (blue) and an activation domain (green)  The DNA binding motif will determine where on the gene the regulator will interact  E.g. Sp1 and CEBP both have different DNA binding motifs which bind different consensus site (maybe part of an enhancer element)  The activator domains can interact with different components of the transcriptional machinery  They will promote the association of the transcription complex (Mediator complex)  The Mediator complex is responsible for interacting on one side with the activators and on the other with the transcriptional machinery o The more interactions made, the higher the likelihood of transcription o Repressors are the opposite; they will have a DNA binding domain and a transcriptional repression domain (which stops formation of the transcription machinery) The Mediator complex is a highly complex piece of machinery with tons of subunits; the naming can become a bit complex:  Subunits corresponding to products of unknown genes are termed MED1, MED2, etc.  Subunits corresponding to the products of yeast genes identified as suppressors of truncations of the CTD of the Pol II large subunit are termed SRBs (for suppressor of RNA Polymerase B) o These genes, when truncated, would make the yeast “ill”; suppressor mutations allowed for the yeast to regain normality o These names were used in older times when the genes weren’t identified  Subunits corresponding to products of genes previously shown to be involved in transcriptional control retain their original names o Examples are TRAPs in human mediator complexes; NUT1, NUT2 in yeast Mediator o Again, these names are older but are kept by convention This is a higher resolution image of the mediator complex (purple) associated with Pol II (yellow)  Remember, in Pol II, the catalytic part of Pol II, beta and beta prime, form a clamp through which the DNA can pass  The mediator complex wraps around the Pol II complex 2 th BIOL 300
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