Fundamentals of Gene Expression-Transcription II PDF

Transcription II [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] TRANSCRIPTION II A. REGULATION OF TRANSCRIPTION IN PROKARYOTES 1. lac Operon Model 2. Basic Components of Operon 3. Negative Regulation of the Operon: lac Repressor 4. Positive Regulation of the Operon by cA...

Transcription II [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] TRANSCRIPTION II A. REGULATION OF TRANSCRIPTION IN PROKARYOTES 1. lac Operon Model 2. Basic Components of Operon 3. Negative Regulation of the Operon: lac Repressor 4. Positive Regulation of the Operon by cAMP B. REGULATION OF TRANSCRIPTION IN EUKARYOTES 1. cis-Acting Regulatory Elements of a Gene 2. trans-Acting Factors 3. Specific Transcription Factors: Binding to cis-Acting Elements 4. Activators (Transactivators) 5. Repressors (Transrepressors) 6. Alternative Mechanisms of Repression 7. Combinatorial Regulation by Transcription Factors 8. Molecular Structure & Function of Specific Transcription Factors C. EPIGENETICS: TRANSCRIPTION AND CHROMATIN STRUCTURE 1. DNA Methylation 2. Covalent Modifications of Histones 3. Histone Code 4. Acetylation of Histones by Histone Acetyltransferases (HATs) 5. Deacetylation of Histones by Histone Deacetyltransferases (HDACs) 6. Noncovalent Remodeling of Chromatin 7. DNase I Hypersensitive Sites D. MOLECULAR MECHANISMS OF TRANSCRIPTIONAL CONTROL 1. Assembly of Pre-initiation Complex on Core Promoter 2. Epigenetic Regulation of Chromatin Structure 3. Regulation by Long Noncoding RNAs (lncRNA) E. INHIBITORS OF TRANSCRIPTION 1. Antibiotics 2. a-Amanitin Suggested Reading Marks’ Basic Medical Biochemistry, 5th Ed: Ch. 16 “What is true for E. coli is also true for the elephant.” Jacques Monod Transcription II [2] OBJECTIVES 1. Specify the major components of the lac operon and describe their corresponding functions in controlling transcription. 2. Describe how lactose stimulates transcription of the lac operon. 3. Explain how cAMP positively regulates transcription in the operon model. 4. Specify the main types of cis-elements in a eukaryotic gene and explain how they function in regulating transcription. 5. Describe the functional differences between general and specific transcription factors. 6. Describe how specific transcription factors bind directly to target genes and explain how they function as either an activator or repressor. 7. Define the concept of combinatorial regulation by transcription factors. 8. Describe 3 mechanisms by which a repressor can inhibit transcription of a target gene. 9. Describe the molecular structure of the bZIP, zinc finger and bHLH families of specific transcription factors and the functions of their respective domains. 10. Define the concept of epigenetics and explain how epigenetic mechanisms regulate transcription of genes by modifying chromatin structure. 11. Define CpG Islands and their functional significance with respect to transcription. 12. Describe how DNA methylation is involved in genomic imprinting. 13. Describe how coactivators and corepressors alter transcriptional activity of a gene. 14. Describe the process of noncovalent chromatin remodeling and its role in regulating transcription. 15. Explain the functional significance of chromatin sites that are hypersensitive to DNase I, 16. Describe how initiation of transcription is regulated through changes in the amount and/or activity of transcription factors. 17. Describe how lncRNAs regulate transcription of target genes. 18. Describe how antibiotics and α-amanitin inhibit transcription. Illustrations adapted from: • Medical Genetics: An Integrated Approach © 2017 McGraw-Hill Education • The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc. • Biochemistry © 2002 by W.H. Freeman and Company Transcription II [3] A. REGULATION OF TRANSCRIPTION IN PROKARYOTES 1. lac Operon Model (Jacob & Monod) An operon is a set of coordinately expressed genes. A single polycistronic mRNA is generated that codes for all of the protein products. Eqtn b-Galactosidase Lactose Glucose + Galactose 2. Basic Components of Operon a) Regulator Gene (i): Codes for repressor or activator protein b) Operator (o): DNA regulatory sequence of the operon c) Operon: Set of coordinately regulated target genes (z, y, a) d) Inducer: Nutrient or metabolite such as lactose i p o z y a 5´ 3´ 3´ 5´ Regulator Gene Promoter Operator Operon 3. Negative Regulation of lac Operon: lac Repressor a) Absence of Lactose: Repressor protein is bound to operator. Transcription of lac operon is repressed because binding of RNA Polymerase to promoter is blocked. i p z y a o 5´ 3´ 3´ 5´ Repressor Protein Repressor is active and binds to the operator. b) Presence of Lactose: Lactose is an inducer that causes a conformational change in the repressor protein such that it becomes inactive and does not bind to operator. i p 5´ 3´ o z y a 3´ 5´ Repressor is inactive. Lactose As a result, transcription of operon is derepressed because promoter is accessible to RNA Polymerase for binding. i 5´ 3´ p o z y a 3´ 5´ Transcription II [4] A. REGULATION OF TRANSCRIPTION IN PROKARYOTES 4. Positive Regulation of lac Operon by cAMP a) Presence of Lactose and Glucose: Transcription of the lac operon does not occur if glucose is still present. This is termed catabolite or glucose repression. Although the repressor has been inactivated by addition of lactose, RNA Polymerase does not bind to the promoter. High glucose inhibits the expression of enzymes involved in breakdown of alternative sugars such as lactose by lowering cAMP. i p z y a o 5´ 3´ 3´ 5´ Lactose Inactive Repressor i p z y a o 5´ 3´ 3´ 5´ b) Presence of Lactose and Absence of Glucose: If glucose is removed, then transcription of the lac operon is induced by a positive control mechanism. [1] Low glucose causes activation of the enzyme Adenylyl cyclase, which increase cAMP levels. [2] cAMP binds to a protein called Catabolite Activator Protein (CAP). [3] CAP•cAMP binds to a consensus sequence near the promoter region of the lac operon. [4] CAP facilitates binding of RNA Polymerase holoenzyme to the promoter of the lac operon to initiate transcription. Low Glucose [1] cAMP • CAP dimer [2] CAP Adenylyl Cyclase cAMP ATP [3] cAMP • i CAP 5´ 3´ p o z y a 3´ 5´ [4] RNA Polymerase holoenzyme cAMP • i 5´ 3´ z CAP y a 3´ 5´ σ lac mRNA (polycistronic) Protein Products: 5´ b-Galactosidase 3´ Permease Transacetylase Transcription II [5] B. REGULATION OF TRANSCRIPTION IN EUKARYOTES 1. cis-Acting Regulatory Elements of a Gene a) Core Promoter: The minimal stretch of DNA that is sufficient to direct accurate initiation of transcription by RNA polymerase II. It encompasses the transcription start site (+1) and ranges between 60-120 bp in length for most genes. Each core promoter contains a combination of consensus DNA sequence elements such as Inr, TATA box, BRE, MTE and DPE. b) Proximal Promoter Elements (PPE): cis-Acting sequences located -200 bp or closer to the transcription start site of the gene. They contain DNA sequences that are binding sites for specific transcription factors that function as either activators or repressors of basal Ex(2) transcription. The CCAAT box and GC box are PPEs found in many genes and are located frequently about 75 to 100 bp upstream of the transcription start site. c) Enhancers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as activators of basal transcription. Enhancers regulate transcription independent of distance from the core promoter and can be located as far as 50 kilobases either upstream or downstream of the transcription start site. Enhancers can still activate transcription when polarity of DNA strands is flipped experimentally in the reverse orientation. d) Silencers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as repressors of transcription. Like enhancers, silencers can function independent of distance from the core promoter and can be located many kilobases either upstream or downstream of the transcription start site. Nota Bene: A single gene is regulated by multiple types of cis-acting elements. Comp(5) Enhancers Silencer core promoter BRE TATA Inr MTE DPE GC CCAAT Box Box 5´ 3´ 3´ 5´ -50 kbp -200 PPE -100 -50 -25 +1 +25 Effect on Basal Transcription Activation PPE Repression Enhancer Activation Upstream Enhancer Activation Reverse Enhancer Activation Downstream Enhancer Activation Upstream/Downstream Silencers Repression Transcription II [6] B. REGULATION OF TRANSCRIPTION IN EUKARYOTES 2. trans-Acting Factors: Proteins that are derived from a gene other than the target gene. transacting factors regulate transcription via cis-acting DNA sequences. • Basal transcription of all genes is dependent on assembly of a pre-initiation complex on the core promoter. In consists of RNA Polymerase II, general transcription factors and the mediator. • Transcription of individual genes is regulated by specific transcription factors that function as activators to increase transcription of target genes above basal levels or as repressors to repress or silence transcription of target genes. Activators and repressors bind directly to DNA sequences in cis-acting elements, but they also have domains for recruiting other transacting factors that function in transcriptional regulation such as coactivators and corepressors. 3. Specific Transcription Factors: Binding to cis-Acting Elements (Don’t memorize this table) Specific Transcription Factor (trans-acting) cis-Acting Element in Target Gene Consensus Binding Sequence in cis-Acting Element GC box GGGCGG Myc, USF E-box CANNTG Activator Protein 1 (AP-1) TRE TGA(G/C)TCA CAAT/Enhancer-binding Protein (C/EBP) CAAT box GGCCAATCT Sp1 Serum Response Factor (SRF) CArG box Estrogen Receptor cAMP Response Element Binding Protein (CREB) CC(A/T)GGG HRE AGGTCANNNTGACCT CRE TGACGTCA N = any nucleotide 4. Activators (Transactivators) Activators are specific transcription factors that have several types of functional domains: a) DNA Binding Domain (DBD): Binds directly to DNA sequences in cis-acting elements of target genes such as PPEs and enhancers. b) Transactivation Domain (TAD): Recruits and/or activates coactivators and other mediator proteins to modify chromatin structure and promote assembly of a pre-initiation complex on the core promoter of a target gene. c) Other Domains: Activators typically have domains for functions such as dimerization, ligand binding (LBD) and nuclear localization (NLS). Many activators also have effector domains for regulation by cell signaling pathways. TAD Coactivator + Fig TAFs DBD B PPE Dimerization Domain RNA Polymerase II Mediator CTD TBP Exon 1 E H A F TAFs TFIID +1 Nota Bene: The same specific transcription factor can regulate transcription of multiple genes that contain the same cis-acting element. Transcription II [7] B. REGULATION OF TRANSCRIPTION IN EUKARYOTES 5. Repressors (Transrepressors) Repressors are specific transcription factors that have several types of functional domains: a) DNA Binding Domain (DBD): Binds directly to cis-acting elements in target genes such as PPEs and silencers. b) Transrepression Domain (TRD): Recruits and/or activates corepressors and other mediator proteins to modify chromatin structure and repress assembly of a pre-initiation complex on the core promoter of a target gene. c) Other Domains: Repressors typically have domains for functions such as dimerization, ligand binding (LBD) and nuclear localization (NLS). Many repressors also have effector domains for regulation by cell signaling pathways. Corepressor TRD RNA Polymerase II Mediator TAFs DBD B PPE CTD TBP Exon 1 E H A F TAFs TFIID Dimerization Domain +1 6. Alternative Mechanisms of Repression a) Block Access to Enhancer Element This type of repressor protein contains a DNA binding domain that binds to a cis-acting element such as an enhancer. Consequently, it blocks access of an activator to the enhancer, thereby repressing transcriptional activity of the gene. Activator Repressor b) Direct Inhibition of Activator Protein This type of repressor protein binds directly to an activator and prevents it from activating transcription. The repressor can inhibit function of the activator by causing a conformational change or through covalent modifications, e.g., phosphorylation/dephosphorylation. Repressor Protein Activator can not dimerize and/or not bind to DNA Repressor Protein Activator binds to DNA, but it can not recruit or interact with coactivators Transcription II [8] r Me dia to Fig B. REGULATION OF TRANSCRIPTION IN EUKARYOTES 7. Combinatorial Regulation by Transcription Factors • Transcription of individual genes is DNA loops can be regulated through combined inter> 50 kb in length! actions of specific transcription Enhancer Activator 2 factors with general transcription factors and RNA Polymerase II. • Looping of DNA facilitates protein Activator 1 interactions between activators RNA and/or repressors and other transPolymerase II PPE cription factors required to assemble TAFs Exon 1 a pre-initiation complex on the core E promoter. Looping enables an enhancer H B TBP A F or silencer to function at great distances TAFs from the core promoter and independently of CTD TFIID +1 orientation relative to the transcription start site. 8. Molecular Structure & Function of Specific Transcription Factors As indicated by this schematic, activators and many repressors for RNA Polymerase II have a DNA binding domain that binds directly to a consensus DNA sequence in a cis-acting element of a target gene such as a PPE, enhancer or silencer (see Table on P. 6). The transactivation or transrepression domain is usually located at or near the N-terminus of the protein. Many types of specific transcription factors function as homodimers or heterodimers, thus, they contain a dimerization domain. Other functional domains may be present such as nuclear localization sequences (NLS), ligand binding domain(s) or Ser/Thr phosphorylation sites. Ser/Thr NLS Ligand binding NTransactivation or Transrepression Domain -C DNA Binding Dimerization Domain domain Specific transcription factors (activators and repressors) are grouped into families based on the molecular structure of the DNA binding domain. a) Basic-Leucine Zipper (bZIP) Family Examples: Activator Protein 1 (AP-1), cAMP Response Element Binding Protein (CREB) This illustration shows that 2 bZIP transcription factors form a dimer for sequence-specific binding to a cis-acting element in DNA. Each cylinder represents the a-helical bZIP domain in the protein. • The leucine zipper holds the 2 proteins of the dimer together like pincers by hydrophobic interactions between the R groups of leucine. • The DNA binding domain is enriched in basic AAs (Lys and Arg) and binds directly to a consensus sequence in a cis-acting element such as an enhancer. Note how the DNA binding domains Nota Bene: Transactivation or transrepression of the dimer fit into the major grooves. domain of bZIP proteins is not shown. B. REGULATION OF TRANSCRIPTION IN EUKARYOTES Transcription II [9] 8. Molecular Structure & Function of Specific Transcription Factors b) Zinc Finger Family Examples: Steroid Hormone Receptors This illustration shows zinc fingers in the DNA binding domain of a steroid hormone receptor. Each zinc finger is composed of 1 a-helix and 2 antiparallel b-sheets stabilized by a zinc ion coordinately bound to 2 Cys and 2 His residues. Note how a zinc finger is positioned in the major groove when binding to DNA. • Binding of a steroid hormone (e.g. estrogen, progesterone, testosterone, glucocorticoid) to Steps(3) the ligand binding domain activates its receptor by causing a conformational change in tertiary protein structure. The activated receptor forms a dimer. • A zinc finger in each DNA binding domain of the receptor dimer binds directly to a specific sequence in the enhancer of a target gene called the hormone response element (HRE). • Once bound to the enhancer, the transactivation domain recruits coactivators (HATs) and other proteins to promote assembly of a pre-initiation complex on the core promoter. Steroid Hormone Dimer Plasma Membrane zinc fingers Nuclear envelope Scheme zinc fingers Activated receptor HRE Dimerization Coactivator RNA Pol II TATA +1 Transcription 5´-A-G-G-T-C-A-N-N-N-T-G-A-C-C-T-3´ 3´-T-C-C-A-G-T-N-N-N-A-C-T-G-G-A-5´ Nota Bene: The DNA binding domain in each half of the dimer binds to the inverted palindromic repeat sequence of HRE in the major grooves. c) Basic Helix-Loop-Helix (bHLH) Family Examples: c-Myc transcription factor, differentiation factors This illustration shows that 2 bHLH transcription factors form a dimer for sequence-specific binding to a cis-acting element in DNA. • The DNA binding domain (larger a-helix) is enriched in basic AAs (Lys and Arg) and binds directly to a consensus E box sequence in a cis-acting element of a target gene. Note how the DNA binding domains of the dimer fit into the major grooves. • The dimerization domain is part of the smaller a-helix. Transcription II [10] C. EPIGENETICS: TRANSCRIPTION AND CHROMATIN STRUCTURE 1. DNA Methylation a) Sequence for Methylation: 5´ CpG 3´ Eqtn CH3 Methyltransferase Cytosine 5-Methylcytosine b) Effects of DNA Methylation: Methylation typically occurs in CpG enriched regions of the genome called CG islands, which are often found in proximity to the core promoter and other regulatory sequence elements of genes. Methylation of DNA decreases and often silences transcription of a gene by epigenetic mechanisms: • DNA methylation can inhibit binding of an activator to cis-acting elements of a target gene. • DNA methylation can promote recruitment of proteins that condense or compact chromatin, for example, HDACs that deacetylate histone tails and/or chromatin remodeling proteins. Thus, chromatin in the promoter region of genes becomes less accessible to transcription factors required for initiation of transcription. c) Genomic Imprinting: Mechanism by which expression of a gene is dependent on whether it is inherited from the paternal or maternal chromosome. Methylation of CpG in either a maternal or paternal gene (allele) occurs during germ cell development. Methylation patterns of genes are maintained by DNA methyltransferases that methylate the daughter strand following DNA replication. In Block 2, Dr. Wolff will cover the effects of genomic imprinting on inheritance patterns for Prader-Willi and Angelman syndromes. CH3 Paternal Gametes 5´ 3´ CG GC 3´ 5´ Methylated allele is inactive CH3 DNA replication during spermatogenesis CH3 5´ 3´ CG GC CH3 5´ 3´ CG GC 3´ 5´ CG GC 5´ 3´ Methylation of daughter strands 3´ 5´ 5´ 3´ CH3 3´ 5´ CH3 CH3 CG GC 3´ 5´ CH3 Maternal 5´ (oocyte) 3´ CG GC Non-methylated allele from mother (active) 3´ 5´ Fertilization generates an inactive & active allele C. EPIGENETICS: TRANSCRIPTION AND CHROMATIN STRUCTURE Transcription II [11] 2. Covalent Modifications of Histones Amino terminal tail Histone N C H2A H2B H3 Ac = Acetylation P = Phosphorylation Me = Methylation Ub = Ubiquitination PubMed - PUbMeAc H4 3. Histone Code: Covalent modifications in the amino terminal tail of histones occur in different combinations to regulate transcription of specific genes. a) Changes in Chromatin Structure: Accessibility of transcription factors to cis-acting elements b) Recruitment of Specific Transcription Factors: Activation or repression of genes 4. Acetylation of Histones by Histone Acetyltransferases (HATs) • HATs acetylate Lys (K) in the amino terminal tails of histones to neutralize positively charged R groups. Coactivator • Relaxes (opens) chromatin structure and makes it more accessible to transcription factors Activator for binding to cis-acting elements in DNA. • Promotes interactions with specific proteins involved in transcription and chromatin remodeling. • HATs often function as a component of coactivator complexes that are recruited to the gene by an activator to acetylate histones. • Generally, increased acetylation of histones in the promoter is associated with gene activation. 5. Deacetylation of Histones by Histone Deacetyltransferases (HDACs) • HDACs deacetylate Lys (K) in amino terminal tails of histones to restore positive charge on R group. Corepressor • Compacts (condenses) chromatin structure and decreases accessibility to transcription factors for binding to DNA sequence elements. Repressor • HDACs often function as part of corepressor complexes that are recruited to the gene by a repressor to deacetylate histones. • Generally, deacetylation of histones in the promoter is associated with transcriptional repression or gene silencing. Acetylation N-terminal tails Deacetylation of N-terminal tails Transcription II [12] C. EPIGENETICS: TRANSCRIPTION AND CHROMATIN STRUCTURE 6. Noncovalent Remodeling of Chromatin a) Remodeling Process: Breaking and reforming of histone-DNA interactions that alter the positioning, shape or number of nucleosomes, thereby affecting accessibility of regulatory elements in genes to transcription factors. b) Chromatin Remodeling Complex: Diverse group of non-histone chromatin proteins that use ATP for energy to unwind DNA from the nucleosome core. They are often recruited to the promoter of genes by specific transcription factors to remodel chromatin. c) Types of Chromatin Remodeling: • Sliding of histone octamers to increase/decrease relative spacing between nucleosomes • Changing conformation of nucleosomes to alter protein interactions • Removal or reassembly of individual nucleosomes Increase spacing in active gene Core Nucleosome Remodeling Factors Histone removal in active gene 7. DNase I Hypersensitive Sites Regions of transcriptionally active euchromatin can be detected by increased sensitivity to DNase I digestion, which produces DNA fragments that are smaller in size than ~ 200 bp protected by nucleosomes in inactive chromatin. Hypersensitive sites result from chromatin remodeling in cis-acting regulatory elements of genes by epigenetic mechanisms. Enhancer of inactive gene DNase I digestion Hypersensitive --> Active DNase I digestion Enhancer of active gene DNase I hypersensitive sites Activator-Coactivator complex Transcription II [13] D. MOLECULAR MECHANISMS OF TRANSCRIPTIONAL CONTROL 1. Assembly of Pre-initiation Complexes on Core Promoter a) Increase or decrease levels of specific transcription factors, i.e., activators, repressors, mediator proteins (coactivators, corepressors) b) Modify activity of transcription factors • Binding to stimulatory or inhibitory ligands • Protein-protein interactions between multiple transcription factors • Covalent modifications such as phosphorylation, acetylation, sumoylation, ubiquitination • Shuttling of transcription factors via nuclear pores 2. Epigenetic Regulation of Chromatin Structure a) DNA methylation b) Histone acetylation and deacetylation c) Chromatin remodeling 3. Regulation by Long Noncoding RNAs (lncRNA) lncRNAs are a class of noncoding RNA that can function similarly to trans-acting proteins by binding to specific DNA sequences, proteins or even other RNAs to regulate gene expression. lncRNAs acquire secondary and tertiary structure that enables them to carry out their functions. The schematic below shows the most well characterized mechanisms by which lncRNAs function in regulating gene expression. a) Decoy lncRNAs: titrate transcription factors and other proteins away from a gene. b) Guide lncRNAs: recruit proteins that modify chromatin structure in target genes, e.g., coactivators, corepressors, chromatin remodeling proteins. c) Scaffold lncRNAs: promote assembly of ribonucleoprotein (RNP) complexes that can remodel chromatin, modify histones or interact with cell signaling components. Adapted from: Cells 8:1178, 2019. E. INHIBITORS OF TRANSCRIPTION 1. Antibiotics a) Actinomycin D: Intercalates into double-stranded DNA and blocks ability of RNA Polymerase to copy the DNA template strand. b) Rifampicin (rifamycin family): Prevents formation of first phosphodiester bond by RNA Polymerase. 2. a-Amanitin: Toxin produced by mushrooms that binds to tightly to RNA Polymerases II to inhibit elongation. It also inhibits RNA Polymerase III at higher concentrations.