Chapter 18: Regulation of Gene Expression

Chapter 18: Regulation of Gene Expression 1. Gene Regulation in Bacteria 2. Gene Regulation in Eukaryotes 3. Gene Regulation in Development 4. Gene Regulation & Cancer

Gene Regulation Gene regulation refers to all aspects of controlling the levels and/or activities of specific gene products. the gene product is either a protein or an RNA molecule regulation can occur at any stage of gene expression which involves accessibility of the gene itself (chromatin structure) transcription & translation (if gene encodes protein) modification of the gene product

Transcription Factors Transcription factors are proteins that either help activate or inhibit transcription. Many transcription factors bind to specific DNA sequences in the regulatory regions of genes. Activation domain DNA DNA-binding domain DNA-binding transcription factors have a DNA-binding domain and one or more activation domains that mediate effects on transcription

1. Gene Regulation in Bacteria Chapter Reading pp. 361-364

Bacterial Gene Regulation Gene regulation in bacteria is generally accomplished at the levels of transcription and post-translational modification of protein activity. Bacterial genes are commonly organized in multi-gene structures called operons: multiple gene coding regions organized in sequence under control of a single promoter genes in the operon are part of same metabolic pathway operons are typically inducible or repressible

Regulation of Tryptophan Production Feedback inhibition Precursor Enzyme 1 trpe gene trpd gene Regulation of gene expression enzymes involved in tryptophan synthesis are part of a single operon (a) Tryptophan Enzyme 2 Enzyme 3 Regulation of enzyme activity trpc gene trpb gene trpa gene (b) Regulation of enzyme production regulation involves transcription & posttranslational modification (feedback inhibition)

Promoter Promoter trp operon Genes of operon DNA Regulatory gene mrna 5 trpr 3 RNA polymerase Operator mrna 5 Start codon trpe trpd trpc trpb trpa Stop codon E D C B A Protein Inactive repressor (a) Tryptophan absent, repressor inactive, operon on DNA mrna No RNA made Polypeptide subunits that make up enzymes for tryptophan synthesis The trp Operon trp repressor is inactive unless bound to tryptophan Protein Tryptophan (corepressor) Active repressor (b) Tryptophan present, repressor active, operon off low tryptophan = ON high tryptophan = OFF repressible operon

DNA mrna Protein Regulatory gene 5 laci 3 Promoter RNA polymerase Active repressor Operator lacz No RNA made (a) Lactose absent, repressor active, operon off The lac Operon lac repressor is active unless bound to allolactose low allolactose = OFF high allolactose = ON lac operon DNA laci lacz lacy laca mrna 5 RNA polymerase 3 mrna 5 Protein -Galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on inducible operon

more on the Promoter lac Operon DNA laci lacz When ON the lac operon is on low by default CAP-binding site camp Active CAP RNA Operator polymerase binds and transcribes If glucose (preferred sugar) is unavailable, lac operon is turned up due to CAP activation camp is produced if glucose is low camp binds and activates CAP active CAP binds CAP site increasing Tx Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (camp level high): abundant lac mrna synthesized DNA laci CAP-binding site Inactive CAP Promoter lacz Operator RNA polymerase less likely to bind Inactive lac repressor (b) Lactose present, glucose present (camp level low): little lac mrna synthesized

2. Gene Regulation in Eukaryotes Chapter Reading pp. 365-376

Overview of Eukaryotic Gene Regulation Eukaryotic genes generally have the following: a single coding region consisting of exons & introns a single promoter multiple proximal and distal control sequences distal control sequences can be 1000s of base pairs away Eukaryotic gene regulation is dependent on chromatin structure in addition all stages between transcription initiation and the production of a functional gene product.

Signal Cap DNA RNA Degradation of mrna Degradation of protein Gene NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Exon Intron Gene available for transcription Transcription Primary transcript RNA processing Tail mrna in nucleus Transport to cytoplasm CYTOPLASM mrna in cytoplasm Translation Polypeptide Protein processing, such as cleavage and chemical modification Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Stages of Gene Regulation Chromatin structure* controls access to genes Transcription key stage of gene regulation RNA processing* splicing of the RNA transcript RNA stability Translation of mrna Post-translation modifications *relevant to eukaryotes only

Chromatin Structure Chromatin structure is regulated through modifications of either the DNA itself or the histone proteins associated with the DNA: DNA modifications addition of methyl (CH 3 ) groups to cytosines results in more compact, less accessible chromatin responsible for X-inactivation, genomic imprinting Histone modifications addition of acetyl groups ( opens chromatin) addition of CH 3 ( closed ) or PO 4 ( open ) groups

Histones & Chromatin Structure Amino acids available for chemical modification Histone tails DNA double helix Nucleosome (end view) (a) Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription DNA is wrapped around histone cores in structures called nucleosomes. tails of histone proteins in nucleosomes can have acetyl, methyl or phosphate groups added to induce a more open or closed chromatin structure

Proximal & Distal Regulation DNA Enhancer (distal control elements) Proximal control elements Transcription start site Poly-A signal sequence Exon Intron Exon Intron Exon Transcription termination region Upstream Distal elements interact with promoter due to bending of DNA. Primary RNA transcript (pre-mrna) mrna Promoter 5 Intron RNA G P P 5 Cap P Transcription Exon Intron Exon Intron Exon 5 UTR Coding segment Start codon RNA processing Stop codon 3 UTR Poly-A signal AAA AAA Poly-A tail Downstream Cleaved 3 end of primary transcript 3 control elements bind specific transcription factors can be located near the promoter (proximal) or very far from the promoter (distal)

DNA Enhancer Current Model of Eukaryotic Transcription Initiation Activators Distal control element DNAbending protein Promoter TATA box General transcription factors Gene Group of mediator proteins Involves specific transcription factors as well as general transcription factors and other proteins involved in all transcription Initiation. Transcription initiation complex RNA polymerase II RNA polymerase II RNA synthesis

Differential Gene Expression Different genes are expressed in different cell types due to: differences in transcription factors differences in chromatin structure Control elements Available activators (a) Liver cell Enhancer LIVER CELL NUCLEUS Albumin gene expressed Crystallin gene not expressed Promoter Albumin gene (b) Lens cell Crystallin gene Available activators LENS CELL NUCLEUS Albumin gene not expressed Crystallin gene expressed

Regulatory roles of non-coding RNA Spliceosomes contain snrna molecules that direct the process of splicing introns from primary RNA transcripts MicroRNAs (mirna) complex with specific proteins to facilitate destruction of specific mrna molecules that contain sequences complementary to mirna sequence target chromatin modification to the centromeres of chromosomes resulting in highly condensed heterochromatin in the centromeres protection from infection by RNA viruses

Alternative Splicing of RNA Exons DNA 1 2 3 4 5 Troponin T gene Primary RNA transcript 1 2 3 4 5 RNA splicing mrna 1 2 3 5 1 2 4 5 or

Hairpin mirna Hydrogen bond Dicer 5 3 (a) Primary mirna transcript mirna mirna mirnaprotein complex Production mrna degraded Translation blocked (b) Generation and function of mirnas

Protein Degradation Ubiquitin Proteasome Proteasome and ubiquitin to be recycled Protein to be degraded Ubiquitinated protein Protein entering a proteasome Protein fragments (peptides) proteins to be degraded in cells (e.g., cyclins) are tagged with a small protein called ubiquitin ubiquitinated proteins are directed to proteosomes which then degrade them

Chromatin modification Genes in highly compacted chromatin are generally not transcribed. Histone acetylation seems to loosen chromatin structure, enhancing transcription. DNA methylation generally reduces transcription. Chromatin modification mrna degradation Transcription RNA processing Translation Protein processing and degradation mrna degradation Each mrna has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Transcription Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Coordinate regulation: Enhancer for liver-specific genes RNA processing Alternative RNA splicing: Primary RNA transcript mrna Enhancer for lens-specific genes Initiation of translation can be controlled via regulation of initiation factors. or Translation Protein processing and degradation Protein processing and degradation by proteasomes are subject to regulation. Summary of Eukaryotic Gene Regulation

3. Gene Regulation in Development Chapter Reading pp. 376-382

Embryonic Development From fertilization to fully developed organism. Involves regulation of maternal and embryonic gene expression: 1 mm 2 mm (a) Fertilized eggs of a frog (b) Newly hatched tadpole Eye Leg Antenna Wild type Mutant maternal genes involved in packaging the egg during oogenesis (egg production) embryonic genes control development after fertilization Mutations in either maternal or embryonic genes can result in developmental defects

Key Events in Animal Development Oogenesis egg production in the ovary results in essential gene regulatory factors (RNA, protein) being packaged very specifically and unevenly in the developing egg Fertilization triggers translation of maternal mrna and rapid series of mitotic nuclear divisions (cleavage) Gastrulation & Induction cell rearrangement and cell-cell signaling resulting in the differentiation of cells and formation of distinct body structures

Early Development (a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Nucleus (b) Induction by nearby cells Cell-cell communication also induces changes in gene expression. Fertilization Zygote (fertilized egg) Two-celled embryo Mitotic cell division Molecules of two different cytoplasmic determinants Egg is packaged unevenly with regulatory factors that are then partitioned into different cells after fertilization. Early embryo (32 cells) Signal transduction pathway Signal receptor Signaling molecule (inducer) NUCLEUS

Head Thorax Abdomen (a) Adult 0.5 mm 1 Egg developing within ovarian follicle Follicle cell Nucleus Egg Nurse cell BODY AXES Anterior Dorsal Left Ventral Right Posterior 2 Unfertilized egg Depleted nurse cells Fertilization Egg shell Early Drosophila 3 Fertilized egg Laying of egg Development Embryonic development maternal genes determine body axes and early pattern formation 4 Segmented embryo 0.1 mm embryonic genes eventually take over and determine subsequent morphogenesis 5 Larval stage Body segments Hatching (b) Development from egg to larva

Bicoid Determines Anterior End The mutant phenotype named Bicoid results in larva with 2 posteriors and no anterior (NO head!). due to a mutation in the maternal Bicoid gene Bicoid mrna is deposited in the anterior end of all eggs during oogenesis Head T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 Wild-type larva 250 m Tail Tail Tail Bicoid activates anterior gene expression after fertilization A8 A8 A7 A6 A7 Mutant larva (bicoid)

Localization of Bicoid Protein, mrna RESULTS Bicoid is a morphogen of maternal origin Anterior end 100 m Fertilization, translation of bicoid mrna Bicoid mrna in mature unfertilized egg Bicoid mrna is expressed into protein after fertilization Bicoid protein in early embryo Bicoid mrna in mature unfertilized egg This results in a Bicoid morphogen gradient Bicoid protein in early embryo

Nucleus Embryonic precursor cell DNA Master regulatory gene myod OFF Other muscle-specific genes OFF Myoblast (determined) mrna MyoD protein (transcription factor) OFF Specification of vertebrate muscle tissue mrna mrna mrna mrna Part of a muscle fiber (fully differentiated cell) MyoD Another transcription factor Myosin, other muscle proteins, and cell cycle blocking proteins

4. Gene Regulation & Cancer Chapter Reading pp. 383-388

Oncogenes Oncogenes are genes with a role in cell cycle progression that have undergone a mutation that contributes to cancer formation (normal version is called a proto-oncogene). generally due to dominant gain-of-function mutations mutations are of 3 general types: 1) translocation of the gene 2) amplification (duplication) of the gene 3) mutations in the coding or regulatory regions of the gene

More on Oncogenes DNA Proto-oncogene Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene within a control element Point mutation: within the gene New promoter Oncogene Oncogene Normal growthstimulating protein in excess Normal growth-stimulating protein in excess Normal growthstimulating protein in excess Hyperactive or degradationresistant protein mutations that result in excessive expression or function can contribute to cancer

1 Growth factor P P P P P P 3 G protein GTP Ras GTP Ras MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own. 2 Receptor Protein kinases (phosphorylation cascade) Ras is a G protein that is a proto-oncogene. Gain-of-function Ras mutations can trigger signal-independent activation of cell cycle. 4 (a) Cell cycle stimulating pathway 5 Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle NUCLEUS

Tumor Suppressor Genes Tumor Suppressor Genes encode gene products that inhibit cell cycle progression. Mutations in tumor suppressor genes are typically recessive loss-of-function mutations. typically requires 2 mutant alleles (recessive) loss of functional gene product leads to defect in: inhibiting cell cycle progression triggering apoptosis activating DNA repair

2 Protein kinases MUTATION UV light 3 Active form of p53 Defective or missing transcription factor, such as p53, cannot activate transcription. 1 DNA damage in genome DNA Protein that inhibits the cell cycle (b) Cell cycle inhibiting pathway

Cancer Requires Multiple Mutations The multi-step or multi-hit hypothesis. Protein overexpressed EFFECTS OF MUTATIONS Protein absent Colon Cell cycle overstimulated Increased cell division Cell cycle not inhibited 1 Loss of tumorsuppressor gene APC (or other) (c) Effects of mutations 2 Activation of ras oncogene 4 Loss of tumorsuppressor gene p53 Normal colon epithelial cells Colon wall Small benign growth (polyp) 3 Loss of tumorsuppressor gene DCC Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma)

Key Terms for Chapter 18 nucleosome, euchromatin, heterochromatin operon, repressor, operator, repressible, inducible control elements, distal, proximal, enhancer general vs specific transcription factors mediator proteins, DNA bending protein mirna, alternative RNA splicing, Dicer, hairpin oogenesis, cytoplasmic determinants, induction morphogenesis, morphogen, morphogen gradient oncogene, proto-oncogene, tumor suppressor gene gain-of-function, loss-of-function mutations Relevant Chapter Questions 1-11