Gene regulation IIIBiochemistry 302March 1, 2006Translation regulation in bacteria:feedback control of transcripts encoding r-proteins• Negative feedback regulation of certain operon transcripts encoding ribosome proteins– β operon contains genes encoding RNAP subunits– str operon contain genes encoding translational elongation factors• Certain r-proteins possess both rRNA & operon-specific mRNA-binding affinity– Repress translation of operon transcripts when level of r-protein > rRNA– Ensures balanced r-protein and rRNA synthesis Differential binding affinity of L10, S7, S4, L4, and S8 for rRNA (higher) and its owns mRNA transcript (lower) makes this mechanism possible.rRNA synthesis is also regulated by a translation-dependent pathway• Stringent response: regulation coordinated with [amino acid]• Amino acid starvation halts rRNA synthesis by a sequence of events triggered by binding of an uncharged tRNA to A site of ribosome then….– Stringent factor (RelA) binds to ribosome– RelA catalyzes addition of pyrophosphate to 3′ position of GTP → guanosine pentaphosphatethen phosphohydrolase removes one phosphate → guanosine tetraphosphate– ppGpp binds to RNA polymerase and alters promoter selectivity (including seven rRNA operons)cAMP and ppGpp are major cellular second messengers in E. coli.Lehninger Principles of Biochemistry, 4th ed., Ch 28Concept of transcription ground state• Prokaryotes: permissive• Eukaryotes: restricted– DNA structure: chromatin “silencing”– Requirement for site-specific DNA-bindingtrans-activators (typically ~5 or more 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 (binding sites for regulatory factors)– 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, 1997Trans-activators function in a cooperative manner to enhance the efficiency of PIC assembly on specific promoters….but this model assumes an accessible DNA template.RegulatedChromatin: natural physical barrier to transcriptional initiation• Models of PIC assembly based on studies using “naked” DNA• β globin gene cluster (first evidence of remodeling)– Chromatin structure rearranged or loosened during differentiation (embryonic → adult globins)– Appearance of DNase I hyper-sensitive 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 chaperones (needed for nucleosome assembly)Lehninger Principles of Biochemistry, 4th ed., Ch 28Many more HATs and HDACs exist with differing substrate specificities and cell/tissue distribution.Dynamics of histone acetylation and deacetylation affects gene activityBacks, J. and Olsen E. N. (2006) Circ. Res. 98:15-24HATs: 5 families (e.g. p300, CBP, pCAF most well studied)HDACs: 3 classes (18 known human HDACs)TF: Indirect RepressorTF: Indirect ActivatorImportance of reversible acetylation of core histones (generally H3 and H4)Acetylation occurs in highly basic N-terminal domain of core histones. Histone code: methylation, acetylation, phosphorylation, ubiquitinationFig. 28-28Lysineε-N-Acetyl-LysineHAT (HistoneAcetyl-Transferase)HistoneDeacetylasereversible reactionsONCCCCCCN+αβγδεPOOOPO-DNAbackbonebindingno DNAbindingONCCCCCCNεOCCAcetyl-CoACoAP---------------------H4 N-terminusH3 N-terminusAc-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 20A-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+++++++++4914182327Chemistry of acetylation of conserved lysine residues in histones© 2002 Waterborg - UMKCChromatin Immunoprecipitation Assay (ChIP)1. Crosslink Protein-DNA complexes in situ (formaldehyde, 2 Å crosslinker)2. Isolate nuclei and fragment DNA (sonication or digestion)3. Immunoprecipitate with antibody against target nuclear protein and reverse crosslinks4a. Identify protein components ofisolated complexes4b. Identify DNA sequence by PCR, cloning and sequencingWestern blotMonitoring of histone modification & TF association in SMαA locus by ChIPMcDonald, O. G. et al. (2006) J. Clin. Invest. 116: 36-48Chromatin “Density”Ac-H4Ac-H3SRFNucleosome or HMG proteins may also facilitate gene activation via DNA looping Fig. 28-27These models show how trans-activators can function at a distance from the core promoter but adaptor proteins are generally needed too. Lehninger Principles of Biochemistry, 4th ed., Ch 28Where remodeling happens is important!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 (e.g. HATs). – 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 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