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Chapter 18~Regulaton of Gene Expression
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Control of Prokaryotic (Bacterial) Genes
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Bacterial metabolism Bacteria need to respond quickly to changes in their environment if they have enough of a product, need to stop production why? waste of energy to produce more how? stop production of enzymes for synthesis if they find new food/energy source, need to utilize it quickly why? metabolism, growth, reproduction how? start production of enzymes for digestion STOP GO
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Remember Regulating Metabolism?
Feedback inhibition product acts as an allosteric inhibitor of 1st enzyme in tryptophan pathway but this is wasteful production of enzymes = inhibition - - Oh, I remember this from our Metabolism Unit!
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Different way to Regulate Metabolism
Gene regulation instead of blocking enzyme function, block transcription of genes for all enzymes in tryptophan pathway saves energy by not wasting it on unnecessary protein synthesis = inhibition - - - Now, that’s a good idea from a lowly bacterium!
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Gene regulation in bacteria
Cells vary amount of specific enzymes by regulating gene transcription turn genes on or turn genes off turn genes OFF example if bacterium has enough tryptophan then it doesn’t need to make enzymes used to build tryptophan turn genes ON example if bacterium encounters new sugar (energy source), like lactose, then it needs to start making enzymes used to digest lactose STOP Remember: rapid growth generation every ~20 minutes 108 (100 million) colony overnight! Anybody that can put more energy to growth & reproduction takes over the toilet. An individual bacterium, locked into the genome that it has inherited, can cope with environmental fluctuations by exerting metabolic control. First, cells vary the number of specific enzyme molecules by regulating gene expression. Second, cells adjust the activity of enzymes already present (for example, by feedback inhibition). GO
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Bacteria group genes together
Operon genes grouped together with related functions example: all enzymes in a metabolic pathway promoter = RNA polymerase binding site single promoter controls transcription of all genes in operon transcribed as one unit & a single mRNA is made operator = DNA binding site of repressor protein
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So how can these genes be turned off?
Repressor protein binds to DNA at operator site blocking RNA polymerase blocks transcription
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Operon model promoter operator
Operon: operator, promoter & genes they control serve as a model for gene regulation RNA polymerase RNA polymerase repressor TATA gene1 gene2 gene3 gene4 DNA promoter operator 1 2 3 4 mRNA enzyme1 enzyme2 enzyme3 enzyme4 Repressor protein turns off gene by blocking RNA polymerase binding site. repressor = repressor protein
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Repressible operon: tryptophan
Synthesis pathway model When excess tryptophan is present, it binds to tryp repressor protein & triggers repressor to bind to DNA blocks (represses) transcription RNA polymerase RNA polymerase repressor trp TATA gene1 gene2 gene3 gene4 DNA promoter operator 1 2 3 4 mRNA trp trp enzyme1 enzyme2 enzyme3 enzyme4 trp trp trp trp repressor repressor protein trp trp trp tryptophan trp conformational change in repressor protein! repressor tryptophan – repressor protein complex trp
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Tryptophan operon What happens when tryptophan is present?
Don’t need to make tryptophan-building enzymes Tryptophan is allosteric regulator of repressor protein
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Inducible operon: lactose
Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA induces transcription RNA polymerase RNA polymerase repressor TATA lac gene1 gene2 gene3 gene4 DNA promoter operator 1 2 3 4 mRNA enzyme1 enzyme2 enzyme3 enzyme4 repressor repressor protein lactose lac conformational change in repressor protein! repressor lactose – repressor protein complex lac
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Lactose operon What happens when lactose is present?
Need to make lactose-digesting enzymes Lactose is allosteric regulator of repressor protein
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Jacob & Monod: lac Operon
1961 | 1965 Jacob & Monod: lac Operon Francois Jacob & Jacques Monod first to describe operon system coined the phrase “operon” Jacques Monod Francois Jacob
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Operon summary Repressible operon Inducible operon
usually functions in anabolic pathways synthesizing end products when end product is present in excess, cell allocates resources to other uses Inducible operon usually functions in catabolic pathways, digesting nutrients to simpler molecules produce enzymes only when nutrient is available cell avoids making proteins that have nothing to do, cell allocates resources to other uses
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Positive gene control occurs when an activator molecule interacts directly with the genome to switch transcription on. Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates. The cellular metabolism is biased toward the utilization of glucose.
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Positive Gene Regulation
Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription
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Positive Gene Regulation
If glucose levels are low (along with overall energy levels), then cyclic AMP (cAMP) binds to cAMP receptor protein (CRP) which activates transcription. If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP protein has an inactive shape and cannot bind upstream of the lac promotor.
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Control of Eukaryotic Genes
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The BIG Questions… How are genes turned on & off in eukaryotes?
How do cells with the same genes differentiate to perform completely different, specialized functions?
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Evolution of gene regulation
Prokaryotes single-celled evolved to grow & divide rapidly must respond quickly to changes in external environment exploit transient resources Gene regulation turn genes on & off rapidly flexibility & reversibility adjust levels of enzymes for synthesis & digestion prokaryotes use operons to regulate gene transcription, however eukaryotes do not. since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off.
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Evolution of gene regulation
Eukaryotes multicellular evolved to maintain constant internal conditions while facing changing external conditions homeostasis regulate body as a whole growth & development long term processes specialization turn on & off large number of genes must coordinate the body as a whole rather than serve the needs of individual cells Specialization each cell of a multicellular eukaryote expresses only a small fraction of its genes Development different genes needed at different points in life cycle of an organism afterwards need to be turned off permanently Continually responding to organism’s needs homeostasis cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment
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Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. transcription 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation
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1. DNA packing as gene control
Degree of packing of DNA regulates transcription tightly wrapped around histones no transcription genes turned off heterochromatin darker DNA (H) = tightly packed euchromatin lighter DNA (E) = loosely packed H E
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DNA methylation Methylation of DNA blocks transcription factors
no transcription genes turned off attachment of methyl groups (–CH3) to cytosine C = cytosine nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body
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Histone acetylation Acetylation of histones unwinds DNA
loosely wrapped around histones enables transcription genes turned on attachment of acetyl groups (–COCH3) to histones conformational change in histone proteins transcription factors have easier access to genes
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Epigenetic Inheritance
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
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2. Transcription initiation
Control regions on DNA promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors “base” rate of transcription enhancer distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription
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Model for Enhancer action
Enhancer DNA sequences distant control sequences Activator proteins bind to enhancer sequence & stimulates transcription Silencer proteins bind to enhancer sequence & block gene transcription Much of molecular biology research is trying to understand this: the regulation of transcription. Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription. Turning on Gene movie
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Transcription complex
Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator B F E RNA polymerase II A TFIID H Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase
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Promoter Activators Gene DNA Enhancer
Fig Promoter Activators Gene DNA Distal control element Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II Figure 18.9 A model for the action of enhancers and transcription activators RNA polymerase II Transcription initiation complex RNA synthesis
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3. Post-transcriptional control
Alternative RNA splicing variable processing of exons creates a family of proteins
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4. Regulation of mRNA degradation
Life span of mRNA determines amount of protein synthesis mRNA can last from hours to weeks RNA processing movie
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5. Control of translation
Block initiation of translation stage regulatory proteins attach to 5' end of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein Control of translation movie
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6-7. Protein processing & degradation
folding, cleaving, adding sugar groups, targeting for transport Protein degradation ubiquitin tagging proteasome degradation The cell limits the lifetimes of normal proteins by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived. Protein processing movie
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Ubiquitin 1980s | 2004 “Death tag” mark unwanted proteins with a label
76 amino acid polypeptide, ubiquitin labeled proteins are broken down rapidly in "waste disposers" proteasomes Since the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere") Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside
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Proteasome Protein-degrading “machine” cell’s waste disposer
breaks down any proteins into 7-9 amino acid fragments cellular recycling A human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdown play Nobel animation
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Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression
Only a small fraction of DNA codes for proteins, rRNA, and tRNA A significant amount of the genome may be transcribed into noncoding RNAs Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration
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RNA interference NEW! Small interfering RNAs (siRNA)
short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA triggers degradation of mRNA cause gene “silencing” post-transcriptional control turns off gene = no protein produced siRNA
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Action of siRNA siRNA mRNA degraded functionally turns gene off
Hot…Hot new topic in biology Action of siRNA dicer enzyme mRNA for translation siRNA double-stranded miRNA + siRNA breakdown enzyme (RISC) mRNA degraded functionally turns gene off
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Gene Regulation 7 6 protein processing & degradation
1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 5 4 initiation of translation mRNA processing 2 1 initiation of transcription mRNA protection mRNA splicing 4 3
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Molecular Biology of Cancer
Oncogene •cancer-causing genes Proto-oncogene •normal cellular genes How? movement of DNA; chromosome fragments that have rejoined incorrectly amplification; increases the number of copies of proto-oncogenes 3-proto-oncogene point mutation; protein product more active or more resistant to degradation Tumor-suppressor genes •changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)
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Cancers result from a series of genetic changes in a cell lineage
The incidence of cancer increases with age because multiple somatic mutations are required to produce a cancerous cell As in many cancers, the development of colon cancer is gradual
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