Gene regulation IIIBiochemistry 302Concept of transcription ground stateTypes of cis-elements that control transcription initiation in eukaryotesChromatin: natural physical barrier to transcriptional initiationImportance of reversible acetylation of core histones (generally H3 and H4)Chemistry of acetylation of conserved lysine residues in histonesSome nucleosomes may facilitate gene activation via promotion of DNA loopingActivation machinery: trans-activators TAFs, and cofactorsBasic principles underlying regulated transcriptional activation in eukaryotesPromoter structure influences the process of activator-mediated chromatin remodeling and PIC assemblyFunctional interplay among multiple TFs governs gene activation in eukaryotesParadigm of transcriptional activation: Recruitment of Pol II GTF machineryStructural features of eukaryotic transcriptional regulators (modularity)Importance of activation domainsReversing transcriptional activation: Mechanisms to inhibit PIC assemblyGene regulation IIIBiochemistry 302Bob Kelm March 2, 2005Concept of transcription ground state• Prokaryotes: permissive• Eukaryotes: restricted– DNA structure: chromatin “silencing”– Requirement for site-specific DNA-bindingtrans-activators (typically ~5 per gene)– Large multi-component regulatory complexes with cofactors mediating protein-protein interaction– Nucleus: Transcription separated from translation in terms of both space and time Lehninger Principles of Biochemistry, 4th ed., Ch 24Types of cis-elements that control transcription initiation in eukaryotes• Core promoter elements (for positioning RNA Pol II)– TATA box (TATAa/tAa/t, – 25 to –35), GC box in TATA-less genes– Inr, Pyr-rich Initiator element (PyPyANa/tPyPy, +1)– DPE, Downstream Promoter Element (+30, yeast)• Regulatory elements– Promoter-proximal elements (e.g. CpG islands, –100 to –200)– Distal enhancer elements– Insulator/boundary elementsfrom Lodish et al., Molecular Cell Biology, 3rdeditionRNA splicing does occur in yeast too.Nikolov and Burley PNAS 94:15, 1997Chromatin: natural physical barrier to transcriptional initiation• Models of PIC assembly really only valid for naked DNA• β globin gene cluster (first evidence of remodeling)– Chromatin structure rearranged at time of differentiation (embryonic → adult globins)– Appearance of DNase I hypersensitive sites →chromatin remodeling• Chromatin remodeling factors (heterochromatin ' euchromatin or mediators of histone binding)– ATP-dependent remodeling– Nuclear (type A) HATs (histone acetyltransferases) and HDACshistone deacetylases); cytosolic (type B) HATs – Histone chaperonesLehninger Principles of Biochemistry, 4th ed., Ch 28Many more HATs and HDACs exist with differing substrate specificities and cell/tissue distribution.Importance of reversible acetylation of core histones (generally H3 and H4)Fig. 28-28Acetylation occurs in highly basic N-terminal domain of core histones. Histone code: methylation, acetylation, phosphorylation, ubiquitinationChemistry of acetylation of conserved lysine residues in histonesAc-S-G-R-G-K-G-G-K-G-L-G-K-G-G-A-K-R-H-R-K-V-L-R-D-++ ++++++++Ac or MeAcAcAcAc581216 20H4 N-terminusA-R-T-K-Q-T-A-R-K-S-T-G-G-K-A-P-R-K-Q-L-A-T-K-A-A-R-K-S-A-P-MeAc or MeAc Ac Ac Ac+++++++++4914182327H3 N-terminusP---------------------Lysineε-N-Acetyl-LysineHAT (HistoneAcetyl-Transferase)HistoneDeacetylasereversible reactionsONCCCCCCN+αβγδεPOOOPO-DNAbackbonebindingno DNAbindingONCCCCCCNεOCCAcetyl-CoACoA© 2002 Waterborg - UMKCSome nucleosomes may facilitate gene activation via promotion of DNA looping Fig. 28-27Lehninger Principles of Biochemistry, 4th ed., Ch 28These 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 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)– 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. – Some exhibit cell and tissue-restricted expression.Basic principles underlying regulated transcriptional activation in eukaryotes• Assembly of a preinitiationcomplex is the key control point but…..• Some chromatin remodeling occurs to ensure that core promoter is open. • 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 cooperateto control transcription from a single promoter.Carey, M. Cell 92:5-8, 1998Promoter structure influences the process of activator-mediated chromatin remodeling and PIC assemblyBefore Yeast HO gene promoterHuman IFN-βgene promoterHuman α1-AT gene promoterDuringAfterelongationC. J. Fry and C. L. Peterson (2002) Science 295:1847-1848Functional interplay among multiple TFsgoverns gene activation in eukaryotesKuman, M. S and Owens, G. K. Arterioscler Thromb Vasc Biol. 23:737-747, 2003Paradigm of transcriptional activation: Recruitment of Pol II GTF machinery• Activators function by recruiting components of the basal transcription machinery to a promoter.• Direct protein-protein interaction between TF activation domains and components of the GTF machinery are necessary.– Reported Kds range from 10−4to 10−7M.– Network of contactsamong multiple activators & subunits of the RNA PolII GTF machinery result in transcriptional synergy.Parabolic responseSigmoidal responseK = e∆G/−RTCarey, M. Cell 92:5-8, 1998Structural 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
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