Gene regulation III Biochemistry 302. Bob Kelm March 2, 2005

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1 Gene regulation III Biochemistry 302 Bob Kelm March 2, 2005

2 oncept of transcription ground state Prokaryotes: permissive Eukaryotes: restricted DNA structure: chromatin silencing Requirement for sitespecific DNAbinding transactivators (typically ~5 per gene) Large multicomponent regulatory complexes with cofactors mediating proteinprotein interaction Nucleus: Transcription separated from translation in terms of both space and time Lehninger Principles of Biochemistry, 4th ed., h 24

3 Types of ciselements that control transcription initiation in eukaryotes from Lodish et al., Molecular ell Biology, 3 rd edition RNA splicing does occur in yeast too. ore promoter elements (for positioning RNA Pol II) TATA box (TATAa/tAa/t, 25 to 35), G box in TATAless genes Inr, Pyrrich Initiator element (PyPyANa/tPyPy, +1) DPE, Downstream Promoter Element (+30, yeast) Regulatory elements Promoterproximal elements (e.g. pg islands, 100 to 200) Distal enhancer elements Insulator/boundary elements

4 Nikolov and Burley PNAS 94:15, 1997

5 hromatin: natural physical barrier to transcriptional initiation Models of PI assembly really only valid for naked DNA β globin gene cluster (first evidence of remodeling) hromatin structure rearranged at time of differentiation (embryonic adult globins) Appearance of DNase I hypersensitive sites chromatin remodeling hromatin remodeling factors (heterochromatin euchromatin or mediators of histone binding) ATPdependent remodeling Nuclear (type A) HATs (histone acetyltransferases) and HDAs histone deacetylases); cytosolic (type B) HATs Histone chaperones Lehninger Principles of Biochemistry, 4th ed., h 28

6 Many more HATs and HDAs exist with differing substrate specificities and cell/tissue distribution.

7 Importance of reversible acetylation of core histones (generally H3 and H4) Fig Acetylation occurs in highly basic Nterminal domain of core histones. Histone code: methylation, acetylation, phosphorylation, ubiquitination

8 hemistry of acetylation of conserved lysine residues in histones H4 Nterminus H3 Nterminus Ac Ac Ac Ac Ac or Me AcSGRGKGGKGLGKGGAKRHRKVLRD Ac or Me Me Ac Ac Ac Ac ARTKQTARKSTGGKAPRKQLATKAARKSAP AcetyloA Lysine HAT (Histone AcetylTransferase) oa εnacetyllysine O O N α β reversible reactions N γ δ ε O O N+ P P O O ε N O Histone Deacetylase DNA backbone binding no DNA binding P 2002 Waterborg UMK

9 Some nucleosomes may facilitate gene activation via promotion of DNA looping Fig Lehninger Principles of Biochemistry, 4th ed., h 28 These models show how transactivators can function at a distance from the core promoter but adaptor proteins are generally needed too.

10 Activation machinery: transactivators TAFs, and cofactors Transactivators (Activators) Some bind to ciselements proximal to the TATA box Some bind to ciselements distal to core promoter or transcription start site (either 5 or 3 ) Some exhibit cell and tissuerestricted expression. TAFs (TBPassociated factors) Adaptor proteins function via proteinprotein interaction Allow distal DNAbound activators to interact with GTFs. ofactors (oactivators, usually large proteins) Adaptor proteins function via proteinprotein 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 tissuerestricted expression.

11 Basic principles underlying regulated transcriptional activation in eukaryotes Assembly of a preinitiation complex is the key control point but.. Some chromatin remodeling occurs to ensure that core promoter is open. Transacting factors (TFs) bind to cisacting 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. arey, M. ell 92:58, 1998

12 Promoter structure influences the process of activatormediated chromatin remodeling and PI assembly Before Yeast HO gene promoter During Human IFNβ gene promoter After elongation. J. Fry and. L. Peterson (2002) Science 295: Human α 1 AT gene promoter

13 Functional interplay among multiple TFs governs gene activation in eukaryotes Kuman, M. S and Owens, G. K. Arterioscler Thromb Vasc Biol. 23:737747, 2003

14 Paradigm of transcriptional activation: Recruitment of Pol II GTF machinery Activators function by recruiting components of the basal transcription machinery to a promoter. Direct proteinprotein interaction between TF activation domains and components of the GTF machinery are necessary. Reported K d s range from 10 4 to 10 7 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 arey, M. ell 92:58, 1998

15 Structural features of eukaryotic transcriptional regulators (modularity) Ordered DNAbinding domain Pseudoordered activation domain Modulate proteinprotein interaction Some TFs possess multiple activation domains Flexible intervening region Separate function domains Protease sensitive onsequences of modularity (creation of novel proteins) Swapping of DNAbinding and activation domains Research and drug development tool or repressor Lodish et al. Molecular ell Biology 3 rd edition

16 Importance of activation domains Defined by mutation analysis, no common 3Dstructure Amino acids often found in activation domains Asp and/or Glurich Glutaminerich Prolinerich Serine/threoninerich Responsible for mediating proteinprotein interaction with cofactors (K d 10 4 to 10 7 M) TBPassociated factors (TAFs) hromatin remodeling factors oactivators and corepressors Lehninger Principles of Biochemistry, 4th ed., h 28

17 Reversing transcriptional activation: Mechanisms to inhibit PI assembly How repressors function 1: ompete with activators for DNAbinding site 2: Inhibit activatorcoactivator or activator:gtf interaction 3: Remodel chromatin (HDAs) Note: Activator and Repressor are descriptive terms. Lehninger Principles of Biochemistry, 4th ed., h 28