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Chapter 18 Regulation of Gene Expression Overview: Conducting the Genetic Orchestra • Prokaryotes and eukaryotes alter gene expression in response to their changing environment • In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types Bacteria regulate their gene expression • Feedback mechanisms allow control over metabolism so that cells produce only the products needed at that time (with some limitations) • This metabolic control occurs on two levels: 1. adjusting activity of metabolic enzymes 2. regulating genes that encode metabolic enzymes 1 Regulation of a Metabolic Pathway Feedback inhibition Precursor trpE gene Enzyme 1 In the pathway for tryptophan synthesis, an abundance of tryptophan can both (a) inhibit the activity of the first enzyme in the pathway (feedback inhibition), a rapid response, and (b) repress expression of the genes for all the enzymes needed for the pathway, a longer-term response. Regulation of gene expression trpD gene Enzyme 2 trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity • (b) Regulation of enzyme production In bacteria, genes are often clustered into operons, composed of: 1. an operator, an on-off switch 2. a promoter 3. genes for metabolic enzymes • An operon can be switched off by a protein called a repressor – binds only to the operator trp operon Promoter Promoter Genes of operon (5) DNA Regulatory gene mRNA trpE trpR 3ʹ′ trpD trpC trpB trpA Operator Start codon Stop codon no tryptophan RNA polymerase mRNA 5ʹ′ 5ʹ′ repressor inactive E D C B A Polypeptides (5) that make up Repressor enzymes for tryptophan synthesis (inactive) Tryptophan absent, repressor inactive, operon on Protein operon ON Operons • A repressible operonis one that is usually on; binding of a repressor to the operator shuts off transcription – the trp operon is a repressible operon – repressible enzymes usually function in anabolic pathways • cells suspend production of an end product that is not needed 2 The trp operon repressor active tryptophan operon OFF DNA No RNA made mRNA Active repressor Protein Tryptophan (corepressor) corepressora small molecule that cooperates with a repressor to switch an operon off Tryptophan present, repressor active, operon off Operons • A repressible operonis one that is usually on; binding of a repressor to the operator shuts off transcription – the trp operon is a repressible operon – repressible enzymes usually function in anabolic pathways • cells suspend production of an end product that is not needed • An inducible operonis one that is usually off; a molecule, an inducer, inactivates the repressor & turns on transcription – the classic example of an inducible operon is the lac operon – inducible enzymes usually function in catabolic pathways • cells only produce enzymes when there s a nutrient that needs to be broken down The lac operon Promoter Regulatory gene Operator lacl DNA lacZ No RNA made no lactose 3ʹ′ mRNA 5ʹ′ RNA polymerase repressor active operon OFF Protein Active repressor Lactose absent, repressor active, operon off The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. 3 Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. lac operon 3 genes DNA 3ʹ′ mRNA lacY lacA Permease Transacetylase lacZ lacl RNA polymerase mRNA 5ʹ′ 5ʹ′ β-Galactosidase Protein E.Coli uses 3 enzymes to take up and metabolize lactose Inactive repressor Allolactose (inducer) β-galactosidase: hydrolyzes lactose to glucose and galactose Lactose present, repressor inactive, operon on permease: transports lactose into the cell transacetylase: function in lactose metabolism is still unclear inducera small molecule that inactivates the repressor repressor inactive lactose operon ON Gene Regulation • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor • Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) • The lac operon is under dual control: – negative control by the lac repressor – positive control by CAP Positive Control • When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP Promoter DNA glucose lacl lacZ CAP-binding site cAMP Active CAP cAMP Inactive CAP RNA Operator polymerase can bind and transcribe Inactive lac repressor Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized 4 When glucose levels increase, CAP detaches from the lac operon, turning it off Promoter DNA lacl lacZ CAP-binding site RNA polymerase can t bind Inactive CAP Operator Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized REVIEW Repressible Operon Genes not expressed Genes expressed Promoter Genes Operator Active repressor: corepressor bound Inactive repressor: no corepressor present Corepressor Inducible Operon Genes not expressed Genes expressed Promoter Operator Genes Active repressor: no inducer present Inactive repressor: inducer bound Eukaryotic Genomes • Two features of eukaryotic genomes are a major information-processing challenge: 1. the typical eukaryotic genome is much larger than that of a prokaryotic cell 2. cell specialization limits the expression of many genes to specific cells The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes 5 Differential Gene Expression • Almost all the cells in an organism are genetically identical • Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome • Errors in gene expression can lead to diseases including cancer Gene Expression • Gene expression is regulated at many stages Signal NUCLEUS Chromatin DNA Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation #1 Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing #2 ***** most important control point #3 Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm #4 #5 Translation Degradation of mRNA Polypeptide Protein processing, such as cleavage and chemical modification #6 Degradation of protein #6 Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) • • • Genes within highly packed heterochromatin (highly condensed areas) are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression histone acetylationacetyl groups (-COCH3) are attached to positively charged lysines in histone tails • This process seems to loosen chromatin structure, thereby promoting the initiation of transcription Histones #1 #1 #2 #3 #4 #5 #6 Histone tails DNA double helix Amino acids available for chemical modification Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones Acetylation of histone tails promotes loose chromatin structure that permits transcription 6 #1 DNA Methylation • DNA methylationthe addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation – In genomic imprinting, methylation turns off either the maternal or paternal alleles of certain genes at the start of development #1 Summary- Chromatin Modifications • Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells • The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance • Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery Eukaryotic Gene • #2 & #3 Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins – Control elements & the proteins they bind are critical to the precise regulation of gene expression in different cell types Enhancer (distal control elements) Proximal control elements Exon Intron Exon Intron Exon Intron Poly-A signal Termination sequence region Exon DNA Upstream Promoter Primary RNA transcript 5ʹ′ (pre-mRNA) #1 Transcription Exon #2 #3 #4 Intron RNA #5 #6 Downstream Poly-A signal Intron Exon Cleaved 3ʹ′ end of primary transcript RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment mRNA 3ʹ′ 5ʹ′ Cap 5ʹ′ UTR (untranslated region) Start codon Stop codon Poly-A 3ʹ′ UTR (untranslated tail region) 7 #2 To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors (TFs) – General TFs are essential for the transcription of all protein-coding genes – In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific TFs § proximal control elementsare located close to the promoter Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. § distal control elementsgroups of which are called enhancers, may be far away from a gene or even in an intron activatorspecific TF, a protein that binds to an enhancer & stimulates transcription of a gene A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. repressorspecific TF, inhibit expression of a gene Combinatorial Control of Gene Activation Enhancer Control elements A particular combination of control elements can activate transcription only when the appropriate activator proteins are present #2 Promoter Albumin gene Crystallin gene LENS CELL NUCLEUS LIVER CELL NUCLEUS Available activators Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell Coordinately Controlled Genes #2 • Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elements • These genes can be scattered over different chromosomes, but each has the same combination of control elements • Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes 8 #3-6 Mechanisms of Post-Transcriptional Regulation • Transcription alone does not account for gene expression • More and more examples are being found of regulatory mechanisms that operate at various stages after transcription • Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes Alternative RNA Splicing #3 Exons DNA Troponin T gene Primary RNA transcript different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons/introns mRNA RNA splicing or mRNA Degradation #4 • The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis • Eukaryotic mRNA is more long lived than prokaryotic mRNA • Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule 9 #5 Initiation of Translation • The initiation of translation of selected mRNAs – can be blocked by regulatory proteins that bind to specific sequences or structures of the mRNA • Alternatively, translation of all mRNAs in a cell may be regulated simultaneously – For example, translation initiation factors are simultaneously activated in an egg following fertilization Protein Processing and Degradation • #6 After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control #1 proteasomesare giant protein complexes that bind protein molecules and degrade them #2 #3 #4 #5 Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol. The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. #6 Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Protein entering a proteasome REVIEW Transcription 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. Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. • 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 Enhancer for liver-specific genes lens-specific genes Chromatin modification Transcription RNA processing RNA processing • Alternative RNA splicing: Primary RNA transcript mRNA degradation Translation Protein processing and degradation mRNA or Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5ʹ′ and 3ʹ′ UTRs. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation. 10 Noncoding RNAs play multiple roles in controlling gene expression • Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA • A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs) • Noncoding RNAs regulate gene expression at two points: • mRNA translation • chromatin configuration Hairpin Hydrogen bond miRNA Dicer 5ʹ′ 3ʹ′ (a) Primary miRNA transcript miRNA miRNAprotein complex • MicroRNAs (miRNAs)small single-stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation mRNA degraded Translation blocked (b) Generation and function of miRNAs • The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) • RNAi is caused by small interfering RNAs (siRNAs) • siRNAs and miRNAs are similar but form from different RNA precursors • In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome • RNA-based mechanisms may also block transcription of single genes 11 REVIEW Chromatin modification Chromatin modification • Small or large noncoding RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Transcription RNA processing mRNA degradation Translation • miRNA or siRNA can block the translation of specific mRNAs. Translation Protein processing and degradation mRNA degradation • miRNA or siRNA can target specific mRNAs for destruction. 12