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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.

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Presentation on theme: "Chapter 18 Regulation of Gene Expression. Overview: Conducting the Genetic Orchestra Prokaryotes and eukaryotes alter gene expression in response to their."— Presentation transcript:

1 Chapter 18 Regulation of Gene Expression

2 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

3 Wild type Mutant Eye Antenna Leg

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5 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

6 Regulation of gene expression trpE gene trpD gene trpC gene trpB gene trpA gene (b) Regulation of enzyme production (a)Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Precursor Feedback inhibition 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 a Metabolic Pathway

7 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 Promoter DNA trpR Regulatory gene RNA polymerase mRNA 3 5 Protein Repressor Tryptophan absent, repressor inactive, operon on mRNA 5 trpE trpD trpC trpBtrpA Operator Start codon Stop codon trp operon Genes of operon (5) E Polypeptides (5) that make up enzymes for tryptophan synthesis D C B A operon ON no tryptophan repressor inactive (inactive)

8 Operons A repressible operon- is 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

9 DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor DNA Protein Tryptophan (corepressor) mRNA Active repressor No RNA made corepressor- a small molecule that cooperates with a repressor to switch an operon off The trp operon operon OFF tryptophan repressor active

10 Operons A repressible operon- is 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 operon- is 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

11 DNA lacl Regulatory gene mRNA 5 3 RNA polymerase Protein Active repressor No RNA made lacZ Promoter Operator Lactose absent, repressor active, operon off The lac operon The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. operon OFF no lactose repressor active

12 DNAlacl mRNA 5 3 lac operon Lactose present, repressor inactive, operon on lacZ lacYlacA RNA polymerase mRNA 5 Protein Allolactose (inducer) Inactive repressor  -Galactosidase Permease Transacetylase Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. 3 genes E.Coli uses 3 enzymes to take up and metabolize lactose β-galactosidase: hydrolyzes lactose to glucose and galactose permease: transports lactose into the cell transacetylase: function in lactose metabolism is still unclear operon ON lactose repressor inactive inducer- a small molecule that inactivates the repressor

13 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

14 DNA cAMP lacl CAP-binding site Promoter Active CAP Inactive CAP RNA polymerase can bind and transcribe Operator lacZ Inactive lac repressor Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP Positive Control glucose cAMP

15 DNA lacl CAP-binding site Promoter RNA polymerase can’t bind Operator lacZ Inactive lac repressor Inactive CAP Lactose present, glucose present (cAMP level low): little lac mRNA synthesized When glucose levels increase, CAP detaches from the lac operon, turning it off

16 Promoter Genes Genes not expressed Active repressor: no inducer present Inactive repressor: inducer bound Genes expressed Operator Promoter Genes Genes not expressed Inactive repressor: no corepressor present Corepressor Active repressor: corepressor bound Genes expressed Operator REVIEW Repressible Operon Inducible Operon

17 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

18 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

19 Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation DNA Gene Gene available for transcription RNA Exon Primary transcript Transcription Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradation of mRNA Polypeptide Protein processing, such as cleavage and chemical modification Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Gene Expression Gene expression is regulated at many stages #1 #2 #3 #4 #6 ***** most important control point #5 #6

20 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 acetylation- acetyl groups (-COCH 3 ) are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription Histone tails Amino acids available for chemical modification DNA double helix Histone tails protrude outward from a nucleosome Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones Histones #1 #2 #3 #4 #6 #5

21 DNA Methylation DNA methylation- the 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

22 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 #1

23 Eukaryotic Gene 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 Upstream DNA Promoter ExonIntron ExonIntron Exon Downstream Transcription Poly-A signal sequence Termination region Intron ExonIntron Exon RNA processing: Cap and tail added; introns excised and exons spliced together Poly-A signal Cleaved 3 end of primary transcript 3 Poly-A tail 3 UTR (untranslated region) 5 UTR (untranslated region) Start codon Stop codon Coding segment Intron RNA 5 Cap mRNA Primary RNA transcript(pre-mRNA) 5 Exon #2 & #3 #1 #2 #3 #4 #5 #6

24 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 elements- are located close to the promoter  distal control elements- groups of which are called enhancers, may be far away from a gene or even in an intron activator- specific TF, a protein that binds to an enhancer & stimulates transcription of a gene repressor- specific TF, inhibit expression of a gene #2 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 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.

25 Control elements Enhancer Promoter Albumin gene Crystallin gene LIVER CELL NUCLEUS Available activators Albumin gene expressed Crystallin gene not expressed (a) Liver cell LENS CELL NUCLEUS Available activators Albumin gene not expressed Crystallin gene expressed (b) Lens cell Combinatorial Control of Gene Activation A particular combination of control elements can activate transcription only when the appropriate activator proteins are present #2

26 Coordinately Controlled Genes 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 #2

27 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 #3-6

28 or RNA splicing mRNA Primary RNA transcript Troponin T gene Exons DNA Alternative RNA Splicing different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons/introns #3

29 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

30 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 #5

31 Protein to be degraded Ubiquitinated protein Proteasome Protein entering a proteasome Protein fragments (peptides) Proteasome and ubiquitin to be recycled Ubiquitin Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control proteasomes- are giant protein complexes that bind protein molecules and degrade them #1 #2 #3 #4 #6 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. Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. #5 #6

32 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. mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. 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 Enhancer for lens-specific genes Transcription RNA processing Alternative RNA splicing: Primary RNA transcript mRNAor Initiation of translation can be controlled via regulation of initiation factors. Protein processing and degradation by proteasomes are subject to regulation. Translation Protein processing and degradation Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation REVIEW

33 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

34 (a) Primary miRNA transcript Hairpin miRNA Hydrogen bond Dicer miRNA- protein complex mRNA degradedTranslation blocked (b) Generation and function of miRNAs 5 3 MicroRNAs (miRNAs)- small single-stranded RNA molecules that can bind to mRNA These can degrade mRNA or block its translation

35 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

36 Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation Chromatin modification Translation mRNA degradation miRNA or siRNA can target specific mRNAs for destruction. miRNA or siRNA can block the translation of specific mRNAs. Small or large noncoding RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. REVIEW

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