UVM BIOC 302 - Gene regulation IV (15 pages)

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Gene regulation IV



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Gene regulation IV

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Pages:
15
School:
The University of Vermont
Course:
Bioc 302 - General Biochemistry
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Gene regulation IV Biochemistry 302 March 3 2006 Chromatin remodeling does not always precede PIC assembly for every gene Before Yeast HO gene promoter During Human IFN gene promoter After Human 1 AT gene promoter elongation C J Fry and C L Peterson 2002 Science 295 1847 1848 Nucleosome or HMG proteins may also facilitate gene activation via DNA looping Where remodeling happens is important Fig 28 27 Lehninger Principles of Biochemistry 4th ed Ch 28 These models show how trans activators can function at a distance from the core promoter but adaptor proteins are generally needed too Activation machinery trans activators TAFs and other cofactors Trans activators Activators Some bind to cis elements proximal to the TATA box Some bind to cis elements distal to core promoter or transcription start site either 5 or 3 Some exhibit cell and tissue restricted expression TAFs TBP associated factors TBP TAFs TFIID Adaptor proteins function via protein protein interaction Allow distal DNA bound activators to interact with GTFs Cofactors Coactivators usually large proteins Adaptor proteins function via protein protein interaction Serve to link transcriptional activators to each other to TAFs or other components of the GTF complex Some possess enzymatic activity e g HATs Some exhibit cell and tissue restricted expression Basic principles underlying regulated transcriptional activation in eukaryotes Assembly of a preinitiation complex is the key control point but Some chromatin remodeling must occur to ensure that cis elements both core regulatory are accessible Trans acting factors TFs bind to cis acting regulatory sequences and recruit the RNA Pol II GTF machinery TF binding sites may be close to or far away from transcription start site Multiple TFs cooperate to control transcription from a single promoter Carey M Cell 92 5 8 1998 Functional interplay among multiple TFs governs gene activation in eukaryotes In this case the coactivating factor myocardin exhibits restricted tissue distribution and is responsible for the cell type specific expression of this gene Kumar M S and Owens G K Arterioscler Thromb Vasc Biol 23 737 747 2003 Paradigm of transcriptional activation Recruitment of Pol II GTF machinery Trans activators work by interacting with specific ciselements to recruit the basal transcription machinery to the core promoter Trans activators also recruit chromatin remodeling factors and other coactivators Direct protein protein interaction between TF activation domains and components of the GTF complex are necessary Kd for protein protein interaction range from 10 4 to 10 7 M while sequence specific DNA binding 10 8 to 10 10 M Network of contacts among multiple activators subunits of the RNA Pol II GTF machinery result in transcriptional synergy Parabolic response Sigmoidal response K e G RT Carey M Cell 92 5 8 1998 Some activators do other important things besides recruiting RNA Pol II complex Orphanides G et al 1996 Genes Development 10 2657 2683 Structural features of eukaryotic transcriptional regulators modularity Ordered DNA binding domain Pseudo ordered activation domain Modulate protein protein interaction Some TFs possess multiple activation domains Flexible intervening region Separate function domains Protease sensitive Consequences of modularity creation of novel proteins Swapping of DNA binding and activation domains Research and drug development tool or repressor Lodish et al Molecular Cell Biology 3rd edition Structure Function of activation i e protein protein interaction domains Defined by mutation analysis no common 3D structure Certain amino acids often found in activation domains Asp and or Glu rich Glutamine rich Proline rich Serine threonine rich Responsible for mediating interaction with cofactors Kd 10 4 to 10 7 M TBP associated factors TAFs Chromatin remodeling factors Coactivators and corepressors Lehninger Principles of Biochemistry 4th ed Ch 28 Preventing transcriptional activation Mechanisms to inhibit PIC assembly How repressors function 1 Compete with activators for DNA binding site 2 Inhibit activator coactivator or activator GTF interaction 3 Remodel chromatin in reverse by HDAC interaction recruitment Lehninger Principles of Biochemistry 4th ed Ch 28 Note Activator and Repressor are descriptive terms Cell and promoter context ligand binding disposition of cofactor partners and PTM can influence TF activity Thermodynamics and chemistry underlying protein DNA interaction General themes Steric shape and electrostatic charge compatibility of protein and DNA Entropy driven binding displaces counter ions water High affinity Kd 10 9 M or less Direct Interactions Specific recognition of individual base pairs by Hbonding between amino acids and functional groups van der Waals contacts to thymine methyl groups Electrostatic contacts to phosphate oxygen atoms Indirect Interactions Bridging through water molecules Induced fitting conformational changes in protein DNA complex to promote binding e g TBP Zinc finger motif first described by A Klug and coworkers in 1986 Structure inferred based on aa sequence analysis of Xenopus laevis TFIIIA 37 zinc fingers Crystal structure 2 1 of the first zinc finger of mouse Zif268 C2H2 30 aa is shown Two antiparallel strands followed by an helix folding stabilized by coordination of a zinc ion Zn2 near the base Zinc ion is chelated by 2 histidines from helix and 2 cysteines one from strand one from loop Multiple zinc fingers needed for high affinity interaction with DNA core recognition element GCG base triplet from Pavletich N P and Sabo C O Science 252 809 817 1991 Refined structure of three C Cys 2H His 2 zinc fingers of Zif268 N4 of C N7 O6 of G from C Pabo lab Elrod Erickson M et al Structure 4 1171 1180 1996 wrapping around the major groove Designer Zinc finger nucleases Useful for gene therapy 64 possible 3 base combinations www scripps edu mb barbas zfdesign zfdesignhome php www zincfingers org Figure from J Kaiser Science 310 1894 1896 2005 Normal rate of HR 1 in 106 cells coexpression of zinc finger nuclease increases this to 1 in 103 cells Delivery into cells and off target effects are the big problems with this approach


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