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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 changing environment

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Chapter 18

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    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. Eye Leg Antenna Mutant Wild type

    4. 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

    5. Precursor Feedback inhibition Regulation of a Metabolic Pathway trpE gene Enzyme 1 • In the pathway for tryptophan synthesis, • an abundance of tryptophan can both • inhibit the activity of the first enzyme in the pathway (feedback inhibition), a rapid response, and • repress expression of the genes for all the enzymes needed for the pathway, a longer-term response. Regulation of gene expression trpD gene trpC gene Enzyme 2 trpB gene Enzyme 3 trpA gene Tryptophan • Regulation of • enzyme activity (b) Regulation of enzyme production

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

    7. 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

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

    9. 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

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

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

    12. 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 Gene Regulation

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

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

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

    16. 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

    17. 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

    18. Signal Gene Expression NUCLEUS Chromatin Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation #1 • Gene expression is regulated at many stages DNA Gene availablefor transcription Gene #2 ***** most important control point Transcription Exon RNA Primary transcript Intron #3 RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm #4 #5 Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification #6 #6 Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

    19. 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 (-COCH3) are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription #1 Histones #1 #2 #3 #5 #4 #6 Histone tails DNA double helix Amino acids available for chemical modification Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones Acetylation of histone tails promotes loose chromatin structure that permits transcription

    20. #1 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

    21. #1 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

    22. #2 & #3 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 Poly-A signal sequence Termination region Exon Intron Exon Exon Intron DNA Upstream Downstream Promoter Transcription Poly-A signal Exon Intron Exon Exon Intron Primary RNA transcript (pre-mRNA) Cleaved 3¢ end of primary transcript #1 5¢ #2 RNA processing: Cap and tail added; introns excised and exons spliced together #3 Intron RNA #5 #4 Coding segment #6 mRNA 3¢ Start codon Stop codon 5¢ UTR (untranslated region) 5¢ Cap Poly-A tail 3¢ UTR (untranslated region)

    23. #2 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 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. repressor- specific TF, inhibit expression of a gene

    24. #2 Combinatorial Control of Gene Activation Enhancer Promoter Controlelements Albumin gene • A particular combination of • control elements can activate • transcription only when the • appropriate activator proteins • are present Crystallingene LENS CELLNUCLEUS LIVER CELLNUCLEUS Availableactivators Availableactivators Albumin genenot expressed Albumin geneexpressed Crystallin genenot expressed Crystallin geneexpressed (a) Liver cell (b) Lens cell

    25. #2 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

    26. #3-6 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

    27. #3 Alternative RNA Splicing Exons DNA Troponin T gene Primary RNA transcript different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons/introns RNA splicing or mRNA

    28. #4 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

    29. #5 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

    30. #6 Protein Processing and Degradation • After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control #1 proteasomes- are giant protein complexes that bind protein molecules and degrade them #2 #3 #4 #5 Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 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. #6 Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Protein entering a proteasome

    31. REVIEW Transcription Chromatin modification • Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors. • Genes in highly compactedchromatin are generally nottranscribed. • Histone acetylation seemsto loosen chromatin structure,enhancing transcription. Bending of the DNA enables activators tocontact proteins at the promoter, initiatingtranscription. • DNA methylation generallyreduces transcription. • Coordinate regulation: Enhancer forliver-specific genes Enhancer forlens-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNAtranscript Translation mRNAdegradation mRNA or Protein processingand degradation Translation • Initiation of translation can be controlledvia regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span,determined in part bysequences in the 5 and3 UTRs. Protein processing and degradation • Protein processing anddegradation by proteasomesare subject to regulation.

    32. 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

    33. Hairpin Hydrogenbond miRNA Dicer 5 3 (a) Primary miRNA transcript miRNA miRNA-proteincomplex • MicroRNAs (miRNAs)- • small single-stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation mRNA degraded Translation blocked (b) Generation and function of miRNAs

    34. 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

    35. REVIEW Chromatin modification • Small or large noncoding RNAs canpromote the formation of heterochromatinin certain regions, blocking transcription. Chromatin modification Transcription Translation RNA processing • miRNA or siRNA can block the translationof specific mRNAs. mRNAdegradation Translation Protein processingand degradation mRNA degradation • miRNA or siRNA can target specificmRNAs for destruction.