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CH. 18 Regulation of gene expression
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nucleus Gene Expression:
Process by which DNA directs the synthesis of proteins Prokaryotic Gene Control Bacteria need to respond quickly to changes in their environment. Lack of a makes this very efficient! Transcription and translation happen simultaneously… they are “coupled” Transcription is what gets regulated. nucleus
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Prokaryotic Control of Metabolism
Gene regulation instead of blocking enzyme function, block transcription of genes for ALL enzymes in the pathway saves energy by not wasting it on unnecessary protein synthesis = inhibition - - -
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Gene regulation in bacteria
Cells vary amount of specific enzymes by regulating gene transcription turn genes off or turn genes on 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 a 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 Two types: INDUCIBLE and REPRESSIBLE Structures of an Operon 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 switches the operon on or off, resulting in coordinated regulation of genes
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Operon Model
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How it works: Operon: operator, promoter & genes they control
RNA polymerase RNA polymerase repressor 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 & Inducible Operons
Both are examples of negative gene regulation (repressor protein turns the operon “off”). Binding of a specific repressor protein to the operator shuts off transcription (the repressor is encoded by a separate regulatory gene) Repressible operon- the repressor is active when bound to a corepressor (which is typically an end-product of an pathway) anabolic
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Repressible Operon
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Repressible operon: tryptophan (trp operon)
Synthesis pathway model When excess tryptophan is present, it binds to trp repressor protein & triggers repressor to bind to DNA blocks (represses) transcription RNA polymerase RNA polymerase repressor trp 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 conformational change in repressor protein! trp tryptophan trp repressor tryptophan – repressor protein complex trp
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Tryptophan operon What happens when tryptophan is present?
Doesn’t need to make tryptophan-building enzymes Tryptophan is allosteric regulator of repressor protein
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Inducible Operon catabolic
Binding of an inducer to an active repressor inactivates the repressor and turns on transcription. Inducible enzymes usually function in pathways. catabolic
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Inducible operon: lactose (lac operon)
Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA induces transcription lac RNA polymerase RNA polymerase repressor 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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Positive gene regulation
Operons are said to have positive control when a protein or enzyme can turn them “ON” or enhance their function by making it easier for RNA polymerase to bind to the promoter. Uses a stimulatory activator protein Ex: CAP/cAMP (cyclic AMP) system
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Positive gene regulation
Ex: CAP/cAMP (cyclic AMP) system When lactose is present and glucose is low: cAMP is high cAMP activates Catabolite Activator Protein (CAP) to bind to a site within the promoter to stimulate transcription. Increases the rate of transcription by 100X!!
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Operon Summary Repressible operon
usually functions in anabolic pathways Used for synthesizing end products when end product is present in excess, cell allocates resources to other uses Inducible operon usually functions in catabolic pathways Used for digesting nutrients in to simpler molecules produces 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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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 DNA & same genes differentiate to perform completely different and 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 Prokaryotic 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 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 homeostasis 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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Several points of control
“Decoupling” of transcription and translation allows for more control. 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 How do you fit all that DNA into nucleus?
DNA coiling & folding double helix Nucleosomes chromatin fiber looped domains chromosome nucleosomes “beads on a string” 1st level of DNA packing histone proteins have high proportion of positively charged amino acids (arginine & lysine) bind tightly to negatively charged DNA from DNA double helix to condensed chromosome
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Nucleosomes “Beads on a string” 1st level of DNA packing
8 histone molecules Nucleosomes “Beads on a string” 1st level of DNA packing DNA wrapped around histone proteins 8 protein molecules positively charged amino acids bind tightly to negatively charged DNA
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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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Epigenetics DNA methylation
Methylation of DNA blocks transcription factors attachment of methyl groups (–CH3) to cytosine) no transcription genes turned off nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body Epigenetics
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Epigenetics Histone acetylation Acetylation of histones unwinds DNA
attachment of acetyl groups (–COCH3) to histones conformational change in histone proteins transcription factors have easier access to genes loosely wrapped around histones enables transcription genes turned on Epigenetics
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2. Transcription Initiation
Control regions on DNA Promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors 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 TF Activator proteins bind to enhancer sequence & stimulates transcription TF Silencer proteins bind to enhancer sequence & block gene transcription (block positive effect of activator proteins) 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.
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3. Post-transcriptional control
Alternative RNA splicing variable processing of exons creates different mRNA molecules Proteins manufactured are related Ex: muscle cell producing two proteins actin and myosin
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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 5’GTP cap and 3’poly A tail prevent degradation
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RNA interference Small interfering RNAs (siRNA) & Micro-RNAs (miRNA)
short segments of RNA (21-28 bases) binds to mRNA “death” tag for mRNA triggers degradation of mRNA causes gene “silencing” post-transcriptional control turns off gene = no protein produced siRNA/ miRNA
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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
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6-7. Protein processing & degradation
folding, cleaving, adding sugar groups, targeting for transport Protein degradation ubiquitin tagging (marked for destruction) proteasome degradation (dismantles protein) 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.
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7 Gene Regulation 6 protein processing & degradation 1 & 2. pre-transcription and 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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How can I induce you to ask questions?
Don’t be repressed! How can I induce you to ask questions?
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