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Lecture 8

Lecture 8

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 24 2012 Lecture 8 Dr. Hekimi Recall Gcn5, a HAT which is part of a large multi-protein complex (SAGA or SLIK)  Gcn4, a transcription factor in yeast is able to bind to the UAS through a DNA binding domain and its activation domain can bind a large multi-protein complex including Gcn5  Remember, SAGA is used in the cell under stressful conditions by activating stress-response genes SAGA Structure:  SPT group: interacts with the TATA Box binding protein (TBP)  Ada group: (Ada1, Ada2, Ada3, Ada4/Gcn5) functionally linked to HAT activity  Tra1: Target DNA bound activators (transcription factors) for recruitment to promoters o This is done with the help of the 19S proteasome related protein, an example of how some proteins are able to multitask (it’s easier for the cell to re-use proteins than making many different ones)  TBP associated factors: (TAFs such as Taf5, 6, 9, etc.) o Showing SAGA is like an alternative to TFIID, with some proteins in common  There are also subunits responsible for de-ubiquitination which bind to the nuclear pore, which might be how these complexes get near the nuclear pore to get more efficient mRNA production during stress conditions  Towards the bottom, we have the HAT activity, as well as an ATPase function  The SAGA complex recognizes modifications made by: o Set1 methylase which works on K4 o Snf1 kinase which works on Serine 10  These bring the SAGA’s Gcn5 subunit closer to acetylate various lysines  SAGA and TFIID make overlapping contributions to expression of Pol II transcribed genes o TFIID: 90% of genes o SAGA: 10% of genes, mainly stress-induced There are other nuclear HAT complexes, 5 in yeast, some of which use proteins other than Gcn5 for the HAT activity:  Many HAT complexes have been isolated from other eukaryotes (some are similar, some are different) 1 th BIOL 300 September 24 2012 Lecture 8 Dr. Hekimi  Some of these contain TAF1, one TBD associated factor, as their acetyltransferase subunit, linking GTFs to HAT activity o Even some general TFs we have seen before use a HAT activity to open up the DNA  One subunit of TFIID contains multiple bromodomains that bind to acetylated histones with high affinity and in fact, the central subunit of TFIID, TAF1, contains HAT activity that is specific for H3 and H4 in vitro o It is thought to keep the promoter hyperacetylated during transcription initiation Histone Deacetylases (HDACs) are the opposite of HATs:  Like HATs, they are large multi-subunit complexes  Yeast has two HDACs: HDAC A and HDAC B o In HDAC A, the deacetylase subunit is termed HDAC1 whereas in HDAC B it is Rpd3 (reduced potassium dependence 3)  This is just another example of how the name of a protein and its function are not necessarily related  Proteins are names based on how they were discovered; that often does not mean their name has anything to do with their function  Many other eukaryotic HDACs have also been identified: o E.g. mammalian mSinA and NuRD (Nucleosome Remodelling histone deacetylase) o The same method of discovery that we discussed for HATs is the same for HDACs; most of the proteins were discovered through amino acid homology and DNA sequence homology to other proteins and genes with HDAC function HDAC B is very similar in binding to HAT:  Ume6 is a transcription repressor which binds to the URS1 sequence through a DNA binding domain  Ume6’s repressor domain then binds the Sin3 subunit of the HDAC B while Rpd3 can carries out its HDAC activity to deacetylate nearby histones to condense the DNA o HDAC activity spans about 3 histones around the Sin3 binding site ATP-dependant chromatin remodelling complexes are a completely different type of chromatin modification protein  All of these proteins contain DNA dependant ATPase activity essential for their remodelling activity o In some older literature, HATs and HDACs are also called remodelling complexes but they work in a different way, and therefore we won’t classify them as remodelling proteins  These complexes use the energy of ATP to introduce torsional stress into the DNA wrapped around the nucleosome  Additional subunits affect specificity through interactions with activators, repressors, and histone tails 2 th BIOL 300 September 24 2012 Lecture 8 Dr. Hekimi  They weaken histone/DNA contacts through the torsional stress, causing fluidity in the position and conformation of nucleosomes  Depending on the gene, they may activate or repress transcription o E.g. the SWI/SNF complex , when knocked out, reverses activation of 3% of the genes in the genome, and reverses repression of another 3% of the genes (showing how it can function both ways) This is very difficult to study this in vivo, but in vitro there are some ways to do it  It’s hard to be able to represent torsional stress on DNA through an experiment; one way to do it is to see what effects changing the DNA’s conformation have on the ability of DNAse 1 to bind o Therefore, based on the number and size of fragments you get out of a chromatin sample, you can make a rough estimation of the chromatin’s conformation around the DNAse binding site There are three families of ATP dependent remodelling complexes based on the type of the ATPase used (through homology to the following proteins):  Swi2/Snf2 ATPase of the yeast Swi/Snf (Switch / Sucrose non-fermenting) complex
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