BIMM 100 Lecture 16

BIMM 100 Lecture 16 - Lecture 16: BIMM100 ­Ge1ng started and fine tuning Reading: pages 296 ­319 Transcrip?onal regula?on • 

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Unformatted text preview: Lecture 16: BIMM100 ­Ge1ng started and fine tuning Reading: pages 296 ­319 Transcrip?onal regula?on •  How to iden?fy transcrip?onal regulators (aka transcrip?on factors) •  The regulators are modular –  dis?nct func?onal domains –  a gallery of mo?fs •  Combinatorial control •  Mul?protein complexes –  enhancers –  Pol II ini?a?on complex itself •  The mechanisms of repression and ac?va?on –  revisi?ng chroma?n! Transcrip?on ini?a?on: RNA Pol II needs other factors! Looking at this in vitro… TATA binding protein (TBP) binds the minor groove and bends DNA •  General transcrip?on factors ­ important for transcrip?on of all mRNAs! •  Highly conserved throughout evolu?on •  This is a stepwise process ­ proteins bind sequen?ally TBP is used in these experiments because the protein complex it is aVached to (TFIID) is too difficult to purify! Also, TFIIA is required in vivo. Transcrip?on ini?a?on: RNA Pol II needs other factors! Looking at this in vitro… TFIIH has helicase ac?vity •  Forma?on of preini?a?on complex (in vivo this is 60 ­70 different polypep?des, and is 3MDa!) Transcrip?on ini?a?on: RNA Pol II needs other factors! Looking at this in vitro… •  ATP hydrolysis is required! •  The CTD is phosphorylated, a]er transcrip?on begins. •  All of the TAFs are released (except TBP) •  Elonga?on occurs (as long as there are NTPs present)! The whole complex… Yeast RNA pol II TFIIH recognizes the stalled polymerase during transcrip?on ­coupled repair! People with defects in TFIIH func?on suffer from diseases such as Cockayne’s syndrome and xoderma pigmnetosum Mechanisms of regula?on •  Remember: the in vivo template for transcrip?on is chroma?n –  Chroma?n is comprised of DNA + histones (and other proteins) •  Histones: have lysine ­rich N terminal tails that are subject to modifica?on –  –  –  –  Acetyla?on Methyla?on Phosphoryla?on Ubiqui?na?on •  These modifica?ons affect transcrip?on! –  Deacetyla?on (nega?ve affect) –  Acetyla?on (posi?ve affect) –  Methyla?on (nega?ve affect) Heterochroma?n is correlated with silenced transcrip?on THE KEY! HML or HMR is transcribed and then transferred to the MAT locus ­ this determines the cell’s ma?ng type Cell ma?ng type control is performed by chroma?n modifica?on in this example (the silenced gene is in a condensed chroma?n structure that sterically prevents transcrip?on factors from interac?ng with it) Proof of chroma*n involvement? 1. All genes in this area are silent, even unrelated tRNA genes transcribed by PolIII 2. E. Coli methyltransferase couldn’t methylate sites within the loci (they were inaccessible) 3. Muta?on of histone tails could de ­repress, restoring the methyltransferases accessibility Silencing of genes at yeast telomeres •  Repressor ac?vator protein 1 (RAP1) and silent informa?on regulator (SIR) proteins work together •  SIR2 is a histone deacetylase! SIR2 mutants have INCREASED acetyla?on! •  SIR3 and 4 bind to the deacetylated tails to SIR1 (not shown), binds generate large, to the HML and HMR inaccessible condensed loci to s?mulate this DNA regions ac?vity at genes to be •  This all prevents other silenced! proteins from binding to start transcrip?on! How do we know this? An important assay: chroma?n immunoprecipita?on (ChIP) Used to map the modifica?on status of different regions of the genome. Requires an?bodies that are directed against specifically modified forms of histones Can also be used to map occupancy of proteins on DNA. This process requires an?bodies directed towards other proteins of interest! ChIP reveals the modifica?on state of par?cular genomic regions 0. Crosslink chroma?n to proteins 1.  Isolate and shear chromosomes so they are about 2 ­3 nucleosomes 2.  Add a specific an?body (in this example it is towards an acetylated histone) 3.  Immunoprecipitate (bind the an?body, which is aVached to the nucleosomes, to a column) 4.  Elute the an?body ­ protein ­DNA complex 5.  Release the DNA from the an?body and protein. 6.  PCR! An example of a ChIP experiment! Is Histone H4 acetyla?on associated with telomeric DNA? Or ac?ve chroma?n? Use WT and Sir2 ­/ ­ cells. You also need primers to amplify the 1.  telomeric DNA region and 2.  ac?ve chroma?n region! An example of a ChIP experiment! Really important! Is Histone H4 acetyla?on associated with telomeric DNA? Or ac?ve chroma?n? Use WT and Sir2 ­/ ­ cells. You use an an? ­acetylated H4 an?body. You also need primers to amplify the 1.  telomeric DNA region and 2.  ac?ve chroma?n region! M Remember ­ Sir2 is an HDAC ­ if it is mutated, yeast telomeres will not be deacetylated! Input No Ab 1 2 wt  ­/ ­ wt  ­/ ­ wt  ­/ ­ wt  ­/ ­ Transcrip?onal regula?on at ini?a?on •  Posi?ve and nega?ve regula?on •  The regulatory sequences and factors •  The mechanisms: –  Assembly and chroma?n modifica?on •  Applica?on? –  Yeast 2 hybrid system! •  Using transcrip?on factor modules to discover interac?ng proteins! •  Regula?on of transcrip?on factor ac?vity –  Ex: nuclear receptors and hormonal control •  Pol I and Pol III ini?a?on •  Termina?on! Model for the mechanism of acetyla?on effects on chroma?n transcrip?on and repression Repression: UME6 binds to a specific upstream element (URS1) of gene X. It’s repression domain binds to SIN3, part of a complex that contains RPD3, an HDAC Histone tails are deacetylated, inhibi?ng binding of transcrip?on factors at the TATA box! Model for the mechanism of acetyla?on effects on chroma?n transcrip?on and repression Ac?va?on: GCN4 binds to a specific upstream ac?va?on sequence element (UAS) of gene X. It’s ac?va?on domain binds to a HAT complex. Histone tails are acetylated, allowing the binding of transcrip?on factors at the TATA box! These two processes are u?lized by many cell types ­ repression and ac?va?on of many genes occur through similar mechanisms! Mechanisms of regula?on ­ remodeling and co ­ac?vators •  Chroma?n remodeling complexes –  Facilitate ac?va?on and repression •  Subunits with helicase homology •  ATPase ­dependent ac?vi?es –  Ex: SWI/SNF complex •  Other co ­ac?vators s?mulate the ini?a?on complex assembly –  Highly coopera?ve assembly reac?ons •  Ex: mediator complex –  Over 20 subunits! Some interact with Pol II, but others interact with ac?vators. One subunit is a HAT! The mediator complex: required for transcrip?onal s?mula?on by Pol II •  •  Well conserved! Forms “bridge” between Pol II and an ac?vator bound at an ac?va?on domain! The mediator complex: several modular ac?vators can bind! •  •  This allows the integra?on of signals from mul?ple different ac?vators at a single promoter! Remember NtrC and the σ54 factor? Same idea ­ it can form a huge loop of DNA! The combined mechanisms of regula?on 1.  Chroma?n modifica?on status “sets the stage” 2.  Chroma?n remodeling complex facilitate ac?va?on 3.  Co ­ac?vators s?mulate the assembly of ini?a?on complexes Combining the mechanisms: ordered binding and interac?on that leads to ac?va?on •  A case study: yeast HO gene •  SWI5 ac?vator binds to distant enhancers (1200 ­1400bp away!) •  The co ­ac?vator (SWI/SNF) complex joins in to help decondense the chroma?n (exposes histone tails) •  GCN5 HAT (in the SAGA complex) joins and acetylates histone tails •  SWI/SNF con?nues to decondense the area Combining the mechanisms: ordered binding and interac?on that leads to ac?va?on •  A case study: yeast HO gene •  SWI5 releases, but GCN5 and SWI/SNF are s?ll there (they bind through bromodomains that recognize the acetyla?on) •  SBF then binds several sites in the proximal promoter region, recrui?ng the mediator complex! •  The binding of the mediator complex to RNA pol II and general transcrip?on factors facilitates the forma?on of the pre ­ini?a?on complex! •  Now, transcrip?on can start! Transcrip?onal regula?on at ini?a?on •  Posi?ve and nega?ve regula?on •  The regulatory sequences and factors •  The mechanisms: –  Assembly and chroma?n modifica?on •  Applica?on? –  Yeast 2 hybrid system! •  Using transcrip?on factor modules to discover interac?ng proteins! •  Regula?on of transcrip?on factor ac?vity –  Ex: nuclear receptors and hormonal control •  Pol I and Pol III ini?a?on •  Termina?on! The yeast two ­hybrid system: u?lizing the power of modular transcrip?on factor domains to discover protein interac?ons The yeast two ­hybrid system: u?lizing the power of modular transcrip?on factor domains to discover protein interac?ons Construc?ng your “bait” and “fish” hybrids… In reality, “fish” are unknown… You screen a library! The yeast two ­hybrid system: u?lizing the power of modular transcrip?on factor domains to discover protein interac?ons BAIT FISH The yeast two ­hybrid system: u?lizing the power of modular transcrip?on factor domains to discover protein interac?ons •  Powerful tool for discovery! •  Can analyze the whole genome for interac?ng proteins •  Can perform drug discovery and small molecule library screens! •  Really important ­ these interac?ons don’t need to be yeast genes! They can be your organism of choice! Transcrip?onal regula?on at ini?a?on •  Posi?ve and nega?ve regula?on •  The regulatory sequences and factors •  The mechanisms: –  Assembly and chroma?n modifica?on •  Applica?on? –  Yeast 2 hybrid system! •  Using transcrip?on factor modules to discover interac?ng proteins! •  Regula?on of transcrip?on factor ac?vity –  Ex: nuclear receptors and hormonal control •  Pol I and Pol III ini?a?on •  Termina?on! Introduc?on to hormones •  Hormones: chemicals that are secreted into the bloodstream that act on specific and/or mul?ple ?ssues simultaneously •  Different hormones have different func?ons –  Hydrophilic •  Pep?des •  Bind to cell ­surface receptors ­ they do not enter the cell itself –  Lipophilic •  Cholesterol ­derived •  Diffuse through the cell membrane to bind receptors that are inside of the cell Nuclear hormone receptors •  There are many intracellular hormone receptors that bind to lipophilic hormones. –  Some reside in the nucleus –  Some reside in the cytoplasm and are translocated into the nucleus under certain condi?ons Domains of nuclear hormone receptors •  Ac?va?on domain (AD) –  Region that regulates transcrip?on •  DNA binding domain (DBD) –  Binds to a specific DNA sequence (o]en known as the hormone response element) •  Inverted repeats: bound by homodimeric nuclear receptors •  Direct repeats: bound by heterodimeric nuclear receptors •  Ligand binding domain (LBD) –  Binds to factors that regulate the ac?vity of the hormone receptor Hormone dependent gene ac?va?on by the glucocor?coid receptor •  Under normal condi?ons, the GR is kept in the cytoplasm, bound to an inhibitor (Hsp90) •  When hormone enters the cell, it diffuses in and binds to the LBD, releasing Hsp90 •  Then, the receptor is translocated to the nucleus, and s?mulates target genes! GR hormone ­binding domain placed upstream of GFP shows how the receptor is translocated to the nucleus when hormone is added! Transcrip?onal regula?on at ini?a?on •  Posi?ve and nega?ve regula?on •  The regulatory sequences and factors •  The mechanisms: –  Assembly and chroma?n modifica?on •  Applica?on? –  Yeast 2 hybrid system! •  Using transcrip?on factor modules to discover interac?ng proteins! •  Regula?on of transcrip?on factor ac?vity –  Ex: nuclear receptors and hormonal control •  Pol I and Pol III ini?a?on •  Termina?on! Pol I ini?a?on: important for life, too! •  Starts with binding of the upstream ac?va?ng factor (UAF) •  Surprise: 2 of the subunits are histones! Probably help DNA binding… Pol I ini?a?on: important for life, too! •  Surprise: TATA binding protein is part of core factor (CF) •  Binds to the Pol I and Rrn3p complex to start transcrip?on •  Cri?cal difference between this and Pol II? Ini?a?on does not require ATP hydrolysis! Pol III ini?a?on •  Different depending on the gene being transcribed •  Requires three general transcrip?on factors –  TFIII (A, B, C) •  Surprise: TBP is part of TFIIIB •  Cri?cal difference between this and Pol II? Ini?a?on does not require ATP hydrolysis! Transcrip?onal regula?on at ini?a?on •  Posi?ve and nega?ve regula?on •  The regulatory sequences and factors •  The mechanisms: –  Assembly and chroma?n modifica?on •  Applica?on? –  Yeast 2 hybrid system! •  Using transcrip?on factor modules to discover interac?ng proteins! •  Regula?on of transcrip?on factor ac?vity –  Ex: nuclear receptors and hormonal control •  Pol I and Pol III ini?a?on •  Termina?on! Termina?on! •  Transcrip?on and transla?on are different! –  Transcrip?on: no “STOP” codons –  Transla?on: “STOP” codons •  Several mechanisms exist in prokaryotes •  We will focus on eukaryo?c regula?on –  Different for each polymerase! Eukaryo?c transcrip?onal termina?on •  Pol I and III transcripts have discrete 3’ ends that are generated by cleavage –  But, they have different mechanisms of cleavage •  Pol II transcripts have variable 3’ ends –  Termina?on occurs through coupled cleavage and polyadenyla?on •  An? ­termina?on and promoter ­proximal pausing are further important regulatory features of Pol II transcrip?on Pol I: Pre ­rRNA synthesis requires a specific DNA ­binding termina?on factor 18bp terminator sequence Pol I Pol I 18bp terminator sequence •  Termina?on occurs at a discrete site about 1000bp downstream of what will become the 3’ end of the mature transcript •  The 3’ end of the transcript is generated by post ­transla?onal cleavage Pol III: terminates a]er a U ­encoding series (of the RNA), embedded in a GC rich region of the DNA I II CCGTTTTTTTGCC Pol Pol I II CCGTTTTTTTGCC •  Usually terminates a]er the second U (some transcripts go to the 3rd or 4th •  No addi?onal termina?on factors are required •  Model? The decreased thermal stability of AAAA matching with UUUU inside of the transcrip?on bubble! Pol II: can terminate at mul?ple sites over 0.5  ­ 2kb beyond the polyA signal Pol I I Pol I I AAUAAA 5’cap cleavage •  Termina?on is coupled to 3’ cleavage and polyadenyla?on •  Pol II can have pausing mechanisms, allowing rapid re ­ini?a?on of transcrip?on without delay for the assembly of ini?a?on complexes •  An? ­termina?on can be important Pol II can have pausing mechanisms ­ this allows the rapid re ­ini?a?on of transcrip?on without delay for the assembly of ini?a?on complexes Pol I I +1 Pauses a]er 25nt ­ does not terminate! HSTF: trimeric heat shock transcrip?on factor •  Example? Heat shock genes! Upon stress and denaturing condi?ons HSTF binds promoter ­ proximal sites, s?mulates elonga?on, and facilitates re ­ ini?a?on An? ­termina?on in Pol II transcrip?on of HIV •  HIV tat encodes a small protein, binds to RNA TAR sequences •  TAR has a stem ­loop structure, bound by both Tat and Cyclin T •  Cyclin T interacts with Cdk9, phosphorylates CTD, promo?ng elonga?on –  Without this interac?on, transcrip?on stops a]er ~50 nucleo?des Tat protein is an an? ­termina?on factor (and therefore a great target for therapeu?cs!) Next class? •  Post ­transcrip?onal regula?on! ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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