Chapter 18: Regulation of Gene Expression 1. Gene Regulation in Bacteria 2. Gene Regulation in Eukaryotes 3. 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 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 Factors factors are proteins that either help activate or inhibit transcription. Many transcription factors bind to specific sequences in the regulatory regions of genes. Activation domain -binding domain -binding transcription factors have a -binding domain and one or more activation domains that mediate effects on transcription 1
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 trp operon romoter romoter Genes of operon Regulatory gene 5 trpr 3 trpe trpd trpc trpb trpa Operator Start codon Stop codon polymerase 5 E D C B A rotein Inactive (a) Tryptophan absent, inactive, operon on rotein Tryptophan (co) Active No made (b) Tryptophan present, active, operon off olypeptide subunits that make up enzymes for tryptophan synthesis The trp Operon trp is inactive unless bound to tryptophan low tryptophan = ON high tryptophan = OFF repressible operon 2
Regulation of Tryptophan roduction Feedback inhibition Tryptophan recursor Enzyme 1 Enzyme 2 Enzyme 3 (a) Regulation of enzyme activity trpe gene trpd gene trpc gene trpb gene trpa gene Regulation of gene expression (b) Regulation of enzyme production enzymes involved in tryptophan synthesis are part of a single operon regulation involves transcription & posttranslational modification (feedback inhibition) rotein Regulatory gene 5 laci 3 romoter polymerase Active Operator lacz No made (a) Lactose absent, active, operon off lac operon The lac Operon lac is active unless bound to allolactose low allolactose = OFF high allolactose = ON laci lacz lacy laca 5 polymerase 3 5 rotein -Galactosidase ermease Transacetylase Allolactose Inactive (inducer) (b) Lactose present, inactive, operon on inducible operon more on the lac Operon When ON the lac operon is on low by default If glucose (preferred sugar) is unavailable, lac operon is turned up due to CA activation cam is produced if glucose is low cam binds and activates CA active CA binds CA site increasing Tx laci CA-binding site cam romoter lacz Operator polymerase Active binds and CA transcribes Inactive CA Allolactose Inactive lac (a) Lactose present, glucose scarce (cam level high): abundant lac synthesized laci CA-binding site Inactive CA romoter lacz Operator polymerase less likely to bind Inactive lac (b) Lactose present, glucose present (cam level low): little lac synthesized 3
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 NUCLEUS Chromatin Stages of Gene Regulation Chromatin modification: unpacking involving histone acetylation and demethylation Gene available for transcription Gene Exon rimary transcript Intron processing Tail Cap in nucleus Transport to cytoplasm CYTOLASM in cytoplasm Degradation Translation of olypeptide rotein processing, such as cleavage and chemical modification Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Chromatin structure* controls access to genes key stage of gene regulation processing* splicing of the transcript stability Translation of ost-translation modifications *relevant to eukaryotes only 4
Chromatin Structure Chromatin structure is regulated through modifications of either the itself or the histone proteins associated with the : 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 O 4 ( open ) groups Histones & Chromatin Structure Amino acids available for chemical modification Histone tails 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 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 Enhancer (distal control elements) Upstream roximal & Distal Regulation Distal elements interact with promoter due to bending of. roximal control elements rimary transcript (pre-) start site romoter 5 Intron G 5 Cap oly-a signal sequence Exon Intron Exon Intron Exon Exon Intron Exon Intron Exon 5 UTR Coding segment Start codon processing Stop codon 3 UTR oly-a signal AAA AAA oly-a tail termination region Downstream Cleaved 3 end of primary transcript control elements bind specific transcription factors can be located near the promoter (proximal) or very far from the promoter (distal) 3 5
Enhancer Current Model of Eukaryotic Initiation Involves specific transcription factors as well as general transcription factors and other proteins involved in all transcription Initiation. Activators Distal control element bending protein initiation complex romoter Gene TATA box General transcription factors Group of mediator proteins polymerase II polymerase II 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 romoter Albumin gene (b) Lens cell Crystallin gene LENS CELL NUCLEUS Available activators Albumin gene not expressed Crystallin gene expressed Regulatory roles of non-coding Spliceosomes contain sn molecules that direct the process of splicing introns from primary transcripts Micros (mi) complex with specific proteins to facilitate destruction of specific molecules that contain sequences complementary to mi sequence target chromatin modification to the centromeres of chromosomes resulting in highly condensed heterochromatin in the centromeres protection from infection by viruses 6
Alternative Splicing of Exons 1 2 3 4 5 Troponin T gene rimary transcript 1 2 3 4 5 splicing 1 2 3 5 or 1 2 4 5 Hairpin mi Hydrogen bond Dicer 5 3 (a) rimary mi transcript mi roduction mi miprotein complex degraded Translation blocked (b) Generation and function of mis rotein Degradation Ubiquitin roteasome roteasome and ubiquitin to be recycled rotein to be degraded Ubiquitinated protein rotein entering a proteasome rotein 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 7
Chromatin modification Genes in highly compacted chromatin are generally not transcribed. Histone acetylation seems to loosen chromatin structure, enhancing transcription. methylation generally reduces transcription. Chromatin modification Regulation of transcription initiation: control elements in enhancers bind specific transcription factors. Bending of the enables activators to contact proteins at the promoter, initiating transcription. Coordinate regulation: Enhancer for Enhancer for liver-specific genes lens-specific genes Summary of Eukaryotic Gene Regulation processing processing Alternative splicing: degradation Translation rimary transcript or rotein processing and degradation Translation degradation Initiation of translation can be controlled via regulation of initiation factors. Each has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. rotein processing and degradation rotein processing and degradation by proteasomes are subject to regulation. 3. 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 8
More on Oncogenes roto-oncogene Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene within a control element oint 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 3 G protein Ras GT GT Ras MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own. 2 Receptor rotein 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 factor (activator) Gene expression rotein 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 repair 9
UV light 2 rotein kinases 3 Active form of p53 MUTATION Defective or missing transcription factor, such as p53, cannot activate transcription. 1 damage in genome rotein that inhibits the cell cycle (b) Cell cycle inhibiting pathway Cancer Requires Multiple Mutations The multi-step or multi-hit hypothesis. rotein overexpressed EFFECTS OF MUTATIONS rotein absent Colon Cell cycle overstimulated Increased cell division Cell cycle not inhibited (c) Effects of mutations 1 Loss of tumorsuppressor 2 Activation gene AC of ras (or other) 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,, operator, repressible, inducible control elements, distal, proximal, enhancer general vs specific transcription factors mediator proteins, bending protein mi, alternative 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 10