biochemistry Regulation of gene expression
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The document describes regulation of gene expression in prokaryotic and eukaryotic cells. In prokaryotes, gene expression is regulated through operons, clusters of genes that are coordinately controlled. The lac and trp operons in E. coli are discussed as examples, with the lac operon being inducible and the trp operon being repressible. In eukaryotes, gene expression can be regulated at many stages including chromatin modifications, transcription, RNA processing, translation and protein modification. This allows for cell specialization and differential gene expression with the same genome.
![1. Chromatin changes
• Heterochromatin is highly condensed that is why
transcriptional enzymes can not reach the DNA
• Acetylation / deacetylation of histons
• Methylation [cytosin] - inactive DNA is highly
methylated
DNA methylation and histone de-acetylation repress
the transcription.](https://image.slidesharecdn.com/u2i4qjes5omfvsmhuo6y-signature-091447bade647bc70325d98b994f7c3a3901d946a34a4516654ab9d79d1d0b85-poli-141026005447-conversion-gate02/85/biochemistry-Regulation-of-gene-expression-26-320.jpg)
![7. Cleavage, chemical modifications
Cleavage
Post-translational modifications
Regulatory proteins [products] are activated
or inactivated by the reversible addition of
phosphate groups / phosphorylation
Sugars on surface of the cell / Glycosylation](https://image.slidesharecdn.com/u2i4qjes5omfvsmhuo6y-signature-091447bade647bc70325d98b994f7c3a3901d946a34a4516654ab9d79d1d0b85-poli-141026005447-conversion-gate02/85/biochemistry-Regulation-of-gene-expression-50-320.jpg)
biochemistry Regulation of gene expression
- 1. Lecture 26 Regulation of gene expression Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation DNA Gene Gene available for transcription Transcription RNA Exon Primary transcript Intron Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation Translation 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)
- 2. Overview • 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 • RNA molecules play many roles in regulating gene expression in eukaryotes
- 3. Regulation of gene expression in prokaryotic cell - Operon units, system of negative feedback in eukaryotic cell come at any stage of gene expression and proteosynthesis. Important are noncoding RNAs.
- 4. Bacteria often respond to environmental change by regulating transcription • Natural selection has favored bacteria that produce only the products needed by that cell • A cell can regulate the production of enzymes by feedback inhibition or by gene regulation • Gene expression in bacteria is controlled by the operon model
- 5. Precursor Feedback inhibition Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Regulation of enzyme activity Regulation of gene expression Regulation of enzyme production - - trpE gene trpD gene trpC gene trpB gene trpA gene
- 6. Operons: The Basic Concept • A cluster of functionally related genes can be under coordinated control by a single “on-off switch” • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control Operon is a functional unit common in bacteria and phages. Activation and inhibition of transcription are regulated in response of conditions in environment. Prokaryotic genetic information is not divided into introns and exons.
- 7. Operon • is coordinately regulated clusters of genes, which are transcribed into one mRNA (polygenic mRNA) • are genes for particular metabolic pathway and are regulated by common promoter and are ordered on DNA following each other
- 8. Escherichia coli Lac operon, Trp operon – model systems = metabolic pathways of • utilization of lactose gen lacZ, lacY, lacA, catabolic pathway with negative and positive regulation • enzymes for TRP synthesis, anabolic pathway with negative regulation
- 9. • The operon can be switched off by a protein repressor • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase • The repressor can be in an active or inactive form, depending on the presence of other molecules • A corepressor is a molecule that cooperates with a repressor protein to switch an operon off • For example, E. coli can synthesize the amino acid tryptophan
- 10. • By default the trp operon is on and the genes for tryptophan synthesis are transcribed • When tryptophan is present, it binds to the trp repressor protein, which turns the operon off • The repressor is active only in the presence of its corepressor--tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high
- 11. Promoter DNA Regulatory gene mRNA trpR 5¢ 3¢ RNA polymerase Protein Inactive repressor Promoter trp operon Genes of operon Operator Start codon Stop codon mRNA 5¢ trpE trpD trpC trpB trpA E D C B A Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA mRNA Protein Tryptophan (corepressor) Active repressor (b) Tryptophan present, repressor active, operon off No RNA made
- 12. Repressible and Inducible Operons: Two Types of Negative Gene Regulation • 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 • An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription
- 13. • The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose • By itself, the lac repressor is active and switches the lac operon off • A molecule called an inducer inactivates the repressor to turn the lac operon on
- 14. Regulatory gene Promoter Operator DNA lacI lacZ mRNA 5¢ 3¢ (a) Lactose absent, repressor active, operon off lacI RNA polymerase (b) Lactose present, repressor inactive, operon on No RNA made RNA polymerase Active Protein repressor lac operon DNA lacZ lacY lacA mRNA 5¢ 3¢ Protein mRNA 5¢ Inactive repressor Allolactose (inducer) b-Galactosidase Permease Transacetylase
- 15. Lac operon - negative regulation • regulatory gene produces repressor, which binds operator and causes that RNAP is not able to initialize transcription • in the presence of lactose repressor is released from operator. The repressor is changed by inducer / lactose RNA polymerase starts the transcription. In 2-3 minutes the amount of enzymes is increased 1000x
- 16. • Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal • Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor
- 17. 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 (cAMP) • Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription
- 18. Lac operon - positive regulation • In the presence of glucose, E. coli preferentially uses glucose for decomposing. • If is low level of glucosis, the cAMP is increased. • CAP (Catabolite activator protein) in the presence of cAMP attaches promoter and activates the transcription. • CAP is allosteric regulatory protein • When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate
- 19. Promoter DNA lacI lacZ CAP-binding site Operator RNA polymerase binds and transcribes cAMP Active CAP Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Promoter DNA lacI lacZ CAP-binding site Operator RNA polymerase less likely to bind Inactive lac repressor Inactive CAP (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
- 20. Summary each operon consists of • promoter (for RNA polymerase) • operator (for repressor) • several structural genes • terminator repressor = allosteric protein encoded by regulatory gene co-repressor = product molecule inducer = substrate molecule
- 21. Eukaryotic gene expression is regulated at many stages • All organisms must regulate which genes are expressed at any given time • In multicellular organisms regulation of gene expression is essential for cell specialization
- 22. Gene expression of eukaryotic cells • each cell maintains specific program / differential gene expression • one mRNA carries information for one gene (monogennic mRNA) • posttranscription modifications of RNA RNA processing and splicing • regulation system is performed at the several levels = transcription, translation, protein activation + secretion
- 23. 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 • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation DNA Gene Gene available for transcription Transcription RNA Exon Primary transcript Intron Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation Translation 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)
- 24. Many steps at which eucaryotic gene expression can be controlled
- 25. more complicated regulating system • chromatin changes • transcription • processing RNA • transport to cytoplasm • degradation of mRNA • translation • cleavage, chemical modification • protein degradation
- 26. 1. Chromatin changes • Heterochromatin is highly condensed that is why transcriptional enzymes can not reach the DNA • Acetylation / deacetylation of histons • Methylation [cytosin] - inactive DNA is highly methylated DNA methylation and histone de-acetylation repress the transcription.
- 27. • DNA methylation is esential for long-term inactivation of genes during cell differentiation Gene imprinting in mamals • methylation constantly turns off the maternal or the paternal allele of a gene in early development • certain genes are expressed in a parent-of-origin-specific manner Epigenetic inheritance
- 28. Histone Modifications • In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails • This loosens chromatin structure, thereby promoting the initiation of transcription • The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin
- 29. Amino acids available for chemical modification Histone tails DNA 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
- 30. 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 regulates expression of either the maternal or paternal alleles of certain genes at the start of development
- 31. 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
- 32. Regulation of Transcription Initiation • 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
- 33. 2. Transcription Transcription factors: proteins that bind DNA and facilitate or inhibit RNA polymerase to bind. They are a part of transcription initiation complex. general transcription factors for all protein-coding genes specific transcription factors – transcription of particular genes at appropriate time and place - enhancers, activators, inhibitors, repressors
- 34. Organization of a Typical Eukaryotic Gene • Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription • Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types
- 35. Enhancer (distal control elements) DNA Proximal control elements Transcription start site Upstream Promoter Poly-A signal sequence Exon Intron Exon Intron Exon Transcription termination region Poly-ADownstream signal Transcription Exon Intron Exon Intron Exon Cleaved 3¢ end of primary transcript 5¢ Primary RNA transcript (pre-mRNA) Intron RNA RNA processing mRNA Coding segment G P P P AAA ××× AAA 5¢ Cap 5¢ UTR Start codon Stop codon 3¢ UTR 3¢ Poly-A tail
- 36. The Roles of Transcription Factors • To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors • General transcription factors 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 transcription factors
- 37. Enhancers and Specific Transcription Factors • Proximal control elements are located close to the promoter • Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron
- 38. • An activator is a protein that binds to an enhancer and stimulates transcription of a gene • Activators have two domains, one that binds DNA and a second that activates transcription • Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene
- 39. • Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription
- 40. Activators DNA Enhancer Distal control element Promoter Gene TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II RNA synthesis Transcription initiation complex
- 41. Cell-type specific transcription: Genes encoding the enzymes of one metabolic pathway are scattered over the different chromosomes - coordinated control in response of chemical signals from outside the cell. The cell accept signals by receptors. Signal transduction pathways activate transcription activators or repressors.
- 43. 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
- 44. Coordinately Controlled Genes in Eukaryotes • 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
- 45. Nuclear Architecture and Gene Expression • Loops of chromatin extend from individual chromosomes into specific Chromosomes in the sites in the nucleus interphase nucleus Chromosome territory • Loops from different chromosomes may congregate at particular sites, some of which are 10 mm rich in transcription factors and RNA polymerases • These may be areas specialized for a common Chromatin Transcription function loop factory
- 46. Mechanisms of Post-Transcriptional Regulation • Transcription alone does not account for gene expression • Regulatory mechanisms can operate at various stages after transcription • Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes
- 47. 3. Processing RNA • In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Exons DNA 3 4 Troponin T gene Primary RNA transcript 3 4 RNA splicing 1 1 2 2 mRNA or 3 2 2 1 1 4 5 5 5 5
- 48. 4, 5. transport of mRNA / degradation • 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
- 49. 6. Translation At the initiation stage – regulatory proteins bind the 5’ end of the mRNA with the cap. Activation or inactivation of protein factors to initiate translation
- 50. 7. Cleavage, chemical modifications Cleavage Post-translational modifications Regulatory proteins [products] are activated or inactivated by the reversible addition of phosphate groups / phosphorylation Sugars on surface of the cell / Glycosylation
- 51. • Polypeptide chain may be cleaved into two or three pieces • Preproinsulin • Proinsulin - disulfide bridges • Insulin • Secretory protein
- 52. Post-translational modifications Acid/base - act/inact Hydrolysis – localization, act/inact Acetylation - act/inact Phosphorylation - act/inact Prenylation - localization Glycosylation - targeting
- 53. 8. protein degradation • Lifespan of protein is strictly regulated • Proteins are produced and degraded continually in the cell. • Proteins to be degraded are tagged with ubiquitin. • Degradation of proteins marked with ubiquitin occurs at the proteasome.
- 55. 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. Transcription • 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 RNA processing • Alternative RNA splicing: Primary RNA transcript mRNA or Translation • Initiation of translation can be controlled via regulation of initiation factors. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation. Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation
- 56. Example of gene regulation
- 57. RReegguullaattiioonn && DDeevveellooppmmeenntt • hhooxx GGeenneess – CCoonnttrrooll OOrrggaann && TTiissssuuee DDeevveellooppmmeenntt IInn TThhee EEmmbbrryyoo –MMuuttaattiioonnss LLeeaadd TToo MMaajjoorr CChhaannggeess • DDrroossoopphhiillaa WWiitthh LLeeggss IInn PPllaaccee ooff AAnntteennnnaaee
- 59. RReegguullaattiioonn && DDeevveellooppmmeenntt hhooxx GGeenneess PPrreesseenntt IInn AAllll EEuukkaarryyootteess – SShhoowwss CCoommmmoonn AAnncceessttrryy –PPaaxx 66 hhooxx ggeennee • CCoonnttrroollss eeyyee ggrroowwtthh iinn DDrroossoopphhiillaa,, MMiiccee && MMaann • PPaaxx 66 ffrroomm MMoouussee PPllaacceedd IInn KKnneeee DDeevveellooppmmeenntt SSeeqquueennccee OOff DDrroossoopphhiillaa DDeevveellooppeedd IInnttoo EEyyee TTiissssuuee.. CCoommmmoonn AAnncceessttoorr >>660000MM YYeeaarrss AAggoo
- 61. Homeotic mutations transform one body part into another.
- 62. Wild type Mutant Eye Antenna Leg
- 63. The Regulation of Eukaryotic Gene Expression ..using the example of PEPCK
- 64. PEPCK • This is an acronym for an enzyme • PhosphoEnol Pyruvate CarboxyKinase • This enzyme is ONLY regulated by gene expression! • No allosteric activators, covalent modification etc • No activation by cAMP, inhibition by insulin etc
- 65. PEPCK • The enzyme is expressed in liver, kidney, adipose tissue and to a lesser extent in muscle • It is a key enzyme in gluconeogenesis (the synthesis of new glucose, usually from lactate, pyruvate or alanine) and glyceroneogenesis (the synthesis of glycerol, usually from lactate, pyruvate or alanine)
- 66. PEPCK overexpression in muscle • a mouse with PEPCK overexpressed in muscle only. • This mouse was leaner than wild type mice, ran for longer and lived longer! • They were also more aggressive. • The overexpression had switched the muscle fuel usage to fatty acids with little lactate production.
- 67. The Supermouse…. • Eats 60% more food than wild type mice • Weighs 40% less than wild type mice • Can run for >4 h until exhaustion whereas the control littermates stop after only 10 min • Has 2 – 3 fold less adipose tissue
- 68. PEPCK overexpression in adipose tissue • A mouse has the PEPCK enzyme overexpressed in adipose tissue. • The results couldn’t be further from supermouse!
- 69. PEPCK overexpression in fat cells
- 70. PEPCK overexpression in adipose tissue • These mice are obese although metabolically healthy (as measured by glucose tolerance and insulin sensitivity) until you put them on a high fat diet. • Then you see insulin resistance and diabetes emerging.
- 71. PEPCK overexpression in liver • Leads to altered glucose tolerance • Insulin resistance • Increased gluconeogenesis causes increased hepatic glucose production which is released into the blood stream • This caused increased insulin secretion but ultimately insulin resistance.
- 72. PEPCK Knock out in liver • Surprisingly these mice can maintain blood glucose under starvation conditions • They develop liver steatosis (fatty livers) probably because of impaired oxidation of fatty acids • A total PEPCK knock out in all tissues is lethal…mice die within days of birth.
- 73. Why the dramatically different outcome for the mouse when PEPCK is overexpressed in different tissues? It is after all the same enzyme catalysing the same reaction.
- 74. Glyceroneogenesis COOH C CH2 PEPcarboxykinase O COOH O C H2C COOH oxaloacetate OAA PO3 Phosphoenol pyruvate PEP COOH C O Pyruvate Carboxylase LDH CH3 COOH HC OH CH3 NADH NAD+ CO2 Pyruvate Lactate CO2 GDP GTP Alanine
- 75. Glyceroneogenesis Fatty acids H3C CO CH2OPO3 C O CH2OH CH2OPO3 HC OH C O Glyceraldehyde 3-P Dihydroxyacetone phosphate (DHAP) H CH2OPO3 HC OH CH2OH Glycerol 3-P Triglycerides S-CoA PEP
Editor's Notes
- #6: Figure 18.2 Regulation of a metabolic pathway.
- #12: The trp operon in E. coli: regulated synthesis of repressible enzymes.
- #15: The lac operon in E. coli: regulated synthesis of inducible enzymes.
- #20: Positive control of the lac operon by catabolite activator protein (CAP).
- #30: A simple model of histone tails and the effect of histone acetylation.
- #36: A eukaryotic gene and its transcript.
- #41: A model for the action of enhancers and transcription activators.
- #45: Cell type–specific transcription.
- #63: Effects of mutations in Hox genes in Drosophila. (c) Normal body structure. (d) Homeotic mutant (bithorax) in which a segment has developed incorrectly to produce an extra set of wings.
- #64: Abnormal pattern formation in Drosophila.