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  1. Chapter 18 On 2010-10-04 by Yeun-Kyu Jang 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 • RNA molecules play many roles in regulating gene expression in eukaryotes

  3. The genetics of bacteria • Concept 18.1: Bacteria often respond to environmental change by regulating transcription • Natural selection has favored bacteria that produce only the products needed by that cell • Bacterial response to environmental change: An individual bacterium can cope with environmental fluctuations by exerting metabolic control • Regulation of a metabolic pathway  Cells can vary the number of specific enzyme molecules by regulating gene expression • Cells can adjust the activity of enzymes already present (for example, by feedback inhibition) (See Fig. 18.2) •  Gene expression in bacteria is controlled by the operon model

  4. (b) Regulation of enzyme production (a) Regulation of enzyme activity Precursor Feedback inhibition Enzyme 1 Gene 1 Regulation of gene expression Gene 2 Enzyme 2 Gene 3 Enzyme 3 – Gene 4 Enzyme 4 – Gene 5 Enzyme 5 Tryptophan Figure 18.2 Regulation of a metabolic pathway In the pathway for tryptophan synthesis, An abundance of Trpcan 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 long term response Tryptophan synthesis pathway

  5. Operons: The Basic Concept • A cluster of functionally related genes can be under coordinated controlby a single on-off “switch” • The regulatory “switch” is a segment of DNA called an operatorusually positioned within the promoter • An operonis the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

  6. Negative regulation of Repressible operon and inducible operon • Repressible operon: function in anabolicpathway  one that is inhibited when a specific small molecule (corepressor) binds allosterically to a regulatory protein (repressor)  Allosteric binding by an corepressor makes the regulatory protein active, and the operon is off  Ex: trpoperon (see Fig. 18.3) • Inducible operon : function in catabolic pathway  One that is stimulated when a specific small molecule interacts with a regulatory protein • Allosteric binding by an inducer molecule makes the regulatory protein (bound repressor) inactive, and the operon is on  Ex: lac operon (See Fig. 18.4) Anabolic: synthesis of biological molecules using ATP energy Catabolic: Generation of ATP by breaking down complex molecules (glucose) Note: Allosteric regulation: the binding of a regulatory molecule to a protein at one site can affect the function of that protein at a different site

  7. trp operon Promoter RNA polymerase Start codon Stop codon Polypeptides that make up enzymes for tryptophan synthesis The trp operon: regulated synthesis of repressible enzymes  “Operon model” discovered by Francois Jacob and Jacues Monod in 1961 Transcription of clustered genes can be controlled by a single promoter via “on-off switch” mode Promoter Genes of operon trpR trpD trpC trpB trpE trpA DNA Operator Regulatory gene 3 mRNA 5 mRNA 5 C E D B A Inactiverepressor Protein (a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes. (Under normal condition, the operon is always turned on) Figure 18.3 Note: Operator: a segment of DNA functions as on-off switch

  8. (b) Tryptophan present, repressor active, operon off. As tryptophan accumulates, it inhibits its own production by activating the repressor protein. DNA TrpR No RNA made mRNA Active repressor Protein Trprepressor:  allosteric protein (Conversion to active form by Trpbinding됨); Operator has two states of repressor-bound or repressor-free Tryptophan (corepressor)

  9. Promoter Regulatorygene Operator DNA lacl lacZ NoRNAmade RNApolymerase mRNA Activerepressor Protein Figure 18.4 The lac operon: regulated synthesis of inducible enzymes (Drinking milk; lactose in milk may be used for energy source) OFF 3 5 Lactose absent, repressor active, operon off (No uptake of milk). The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. (Under normal condition lacking lactose, bacterial cells do not need the enzymes for lactose utilization, so lac operon is off) (a)

  10. RNApolymerase (b) Lactose present, repressor inactive, operon on (Uptake of milk). Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. ON lac operon DNA lacl lacz lacY lacA 3 mRNA 5 mRNA mRNA 5' 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Used for lactose utilization Allolactose(inducer) This lac operon is not simple like this. To produce abundant mRNA from this operon, another option is demanded

  11. 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 • Inducible operon such as lac operon requires another control mechanism to get optimal gene products : Positive control system

  12. Positive control of inducible operon Positive control  Gene regulation is said to be positive only when a regulatory protein interacts directly with the genome to switch transcription on • Ex: lac operon (See Fig. 18.5) • Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates including glucose  If glucose levels are low, then cyclic AMP (cAMP) accumulates → binds to CAP (catabolite activator protein) → attach directly to the promoter → activate the transcription of lac operon

  13. Figure 18.5 Positive control of the lac operon by catabolite activator protein (CAP) Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can bindand transcribe ActiveCAP cAMP Inactive lac repressor InactiveCAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for the lactose pathway. (a)

  14. (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind InactiveCAP Inactive lac repressor

  15. Concept 18.2: Eukaryotic gene expression can be regulated at any stage • All organisms • Must regulate which genes are expressed at any given time • During development of a multicellular organism • Its cells undergo a process of specialization in form and function called cell differentiation

  16. 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 is regulated at many stages

  17. Signal NUCLEUS • Many key stages of gene expression • Can be regulated in eukaryotic cells • Transcriptional control • Posttranscriptional control • Translational control • Posttranslational control Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Chromatin Chromatin modification DNA Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradation of mRNA Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function Figure 18.6

  18. Regulation of Chromatin Structure • Genes within highly condensed heterochromatin • Are usually not expressed Histone code (Safety system; Dynamic) Genetic code (Black-box; Static)

  19. Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Histone tails DNA double helix Amino acids available for chemical modification Histone Modification • Chemical modification of histone tails • Can affect the configuration of chromatin and thus gene expression Figure 18.7a (a) Histone tails protrude outward from a nucleosome

  20. Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Figure 18.7b • Histone acetylation • Seems to loosen chromatin structure and thereby enhance transcription

  21. In histoneacetylation, acetyl groups are attached to positively charged lysines in histone tails • This process 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 • The histone code hypothesis proposes that specific combinations of modifications help determine chromatin configuration and influence transcription

  22. DNA Methylation • DNA methylation, the addition of methyl groups to certain bases (cytosine) in DNA, is associated with transcriptional repression in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation (70% of CpG are methylated in differentiated somatic cells) • In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

  23. 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

  24. 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

  25. Organization of a Typical Eukaryotic Gene • Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins (transcription factors etc) • Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types

  26. Organization of a Typical Eukaryotic Gene Proximal control elements Poly-A signal sequence Termination region Enhancer (distal control elements) Exon Intron Intron Exon Exon DNA Downstream Upstream Transcription Promoter Poly-A signal Cleared 3 end of primary transport Exon Exon Intron Intron Exon Primary RNA transcript (pre-mRNA) 5 Chromatin changes RNA processing: Cap and tail added; introns excised and exons spliced together Transcription Intron RNA RNA processing Coding segment mRNA degradation Translation mRNA P G Protein processing and degradation P P Start codon Poly-A tail 5 Cap Stop codon 3 UTR (untranslated region) 5 UTR (untranslated region) Figure 18.8: A eukaryotic gene and its transcript

  27. 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

  28. Enhancers and Specific Transcription Factors • Proximal control elements • Are located close to the promoter • Distal control elements, groups of which are calledenhancers • May be far away from a gene or even in an intron

  29. An activator • Is a protein that binds to an enhancer and stimulates transcription of a gene Figure 18.9: A model for the action of enhancers and transcription activators Distal control element Promoter Activators Gene TATA box Enhancer General transcription factors Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 1 DNA-bending protein Group of Mediator proteins A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. 2 RNA Polymerase II The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. Chromatin changes 3 Transcription RNA processing RNA Polymerase II mRNA degradation Translation Protein processing and degradation Transcription Initiation complex RNA synthesis

  30. Some transcription factors function as repressors, inhibiting expression of a particular gene • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription

  31. Coordinately Controlled Genes • Unlike the genes of a prokaryotic operon, each of the coordinately controlled 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

  32. 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

  33. Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript RNA splicing or mRNA RNA Processing • 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 Figure 18.11

  34. 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 • The mRNA life span is determined in part by sequences in the leader and trailer regions

  35. 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 (within 5’-UTR), preventing the attachment of ribosomes • In the eggs, the stored mRNAs lack poly(A) tails required for initiation of translation  an enzyme adds more A residues, allowing translation to begin • Alternatively, translation of all the mRNAs in a cell • May be regulated simultaneously • Ex: Translation of mRNAs stored in egg cells is triggered by the sudden activation of translation initiation factors just after fertilization

  36. Protein Processing and Degradation • After translation • Various types of protein processing, including cleavage and the addition of chemical groups (sugar, phosphate etc), are subject to control • Regulation of the length of time in protein function by selective degradation • (Ex: Cyclin’s half life in control of cell cycle  see Fig. 18.12)

  37. Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 3 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 1 Multiple ubiquitin mol- ecules are attached to a protein by enzymes in the cytosol. Chromatin changes Transcription RNA processing Ubiquitin Proteasome and ubiquitin to be recycled Translation mRNA degradation Proteasome Protein processing and degradation Protein fragments (peptides) Ubiquinated protein Protein to be degraded Protein entering a proteasome • Proteasomes • Are giant protein complexes that bind protein molecules and degrade them Figure 18.12: Degrdation of a protein by a proteasome

  38. 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

  39. Effects on mRNAs by MicroRNAs and Small Interfering RNAs • MicroRNAs(miRNAs) are small single-stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation • 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

  40. Fig. 18-13: Regulation of gene expression by miRNAs Hairpin miRNA  Hydrogen bond  Dicer  miRNA miRNA- protein complex 5 3 (a) Primary miRNA transcript   An enzyme cuts each hairpin from the primary miRNA transcript  A 2nd enzyme Dicer trims the loop and the single-stranded ends from the hairpin, cutting at the arrows  One strand of the double-stranded RNA is degraded; the other strand (miRNA) then forms a complex with one or more proteins   The miRNA in the complex can bind to any target mRNA that contains at least 6 bases of complementary sequence  If miRNA and mRNA bases are complementary all along their length, the mRNA is degraded (left); if the match is less complete, translation is blocked (right) mRNA degraded Translation blocked (b) Generation and function of miRNAs

  41. Chromatin Remodeling and Silencing of Transcription by Small RNAs • siRNAs play a role in heterochromatin formation and can block large regions of the chromosome • Small RNAs may also block transcription of specific genes

  42. Concept 18.4: A program of differential gene expression leads to the different cell types in a multicellular organism • During embryonic development, a fertilized egg gives rise to many different cell types • Cell types are organized successively into tissues, organs, organ systems, and the whole organism • Gene expression orchestrates the developmental programs of animals

  43. A Genetic Program for Embryonic Development • The transformation from zygote to adult results from cell division, cell differentiation, and morphogenesis

  44. Fig. 18-14 From fertilized egg to animal: what a difference four days makes (a) Fertilized eggs of a frog (b) Newly hatched tadpole Cell division, differentiation, and morphogenesis to transform each of the fertilized eggs into a tadpole

  45. Cell differentiation is the process by which cells become specialized in structure and function • The physical processes that give an organism its shape constitute morphogenesis • Differential gene expression results from genes being regulated differently in each cell type • Materials in the egg can set up gene regulation that is carried out as cells divide

  46. Regulation of early development by cytoplasmic determinants and inductive signals • An egg’s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg • Cytoplasmic determinants are maternal substances in the egg that influence early development • As the zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different gene expression

  47. Fig. 18-15a Unfertilized egg cell Q: Sources of developmental information for the early embryo Sperm Nucleus Unevenly distributed determinants in unfertilized egg Fertilization Two different cytoplasmic determinants Zygote After fertilization and mitotic division, the cell nuclei of the embryo are exposed to different sets of determinants, leading to expression of different genes Mitotic cell division Two-celled embryo (a) Cytoplasmic determinants in the egg

  48. Early embryo (32 cells) Signal transduction pathway NUCLEUS Signal receptor Signal molecule (inducer) • The other important source of developmental information is the environment around the cell, especially signals from nearby embryonic cells • In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells • Thus, interactions between cells induce differentiation of specialized cell types Figure 18.15b Induction by nearby cells. The cells at the bottom of the early embryo depicted here are releasing chemicals that signal nearby cells to change their gene expression. (b)

  49. Sequential Regulation of Gene Expression During Cellular Differentiation • Determinationcommits a cell to its final fate • Determination precedes differentiation • Cell differentiation is marked by the production of tissue-specific proteins

  50. Myoblastsproduce muscle-specific proteins and form skeletal muscle cells • MyoDis one of several “master regulatory genes” that produce proteins that commit the cell to becoming skeletal muscle • The MyoD protein is a transcription factor that binds to enhancers of various target genes