Control Of Eukaryotic Gene Expression (Learning Objectives)

Control of Eukaryotic Gene Expression (Learning Objectives)1. Compare and contrast chromatin and chromosome: composition, proteinsinvolved and level of packing. Explain the structure and function of nucleosome,histones, scaffold proteins (metaphase chromosomes)2. Explain the role of non-coding RNA and chemical modifications: methylation ofDNA and acetylation of histones in control of gene expression. Define the termepigenetics.3. Identify the main mechanism for turning on gene expression. Explain why controlof gene expression in eukaryotic cells is like a “dimmer switch”, an “ON” switchthat can be fine tuned.4. Identify the major switch and all the fine-tuning steps that can modulateeukaryotic gene expression.5. Identify and explain component of eukaryotic genes: coding and regulatorysequences (proximal and distal elements)6. Compare and contrast pre and post transcriptional and translational controls ofgene expression7. Explain interference RNA and its role play in post-transcriptional and translationalregulation of gene expression8. Define ubiquitin and proteosome and explain their roles in intracellular proteindegradation

Control of Eukaryotic GeneExpression

Control of Eukaryotic Gene Expression Cells express 3-5% of their genes– House-keeping genes- all the time– Genes turned on or off- internal and externalsignals (environmental- nurture)– Genes turned on only in some cell types notothers while others are permanently shut down(Highly specialized nerves or muscle)

Structural organization of EukaryoticDNA– Nucleosomal beads of chromatin: DNA and histones“Beads on a string”– Chromatin packing is the degree of nucleosome coiling– Histones play major role in gene expressionExpose DNA when it isto be transcribed shieldit when it is to be silencedThe human genome codes for 21,000 genes

InterphaseChromatin nactiveEuchromatin:open, activelytranscribed

Eukaryotic Chromatin StructureSuccessive levels of chromatin packing1.2.3.4.ds-DNA helix (naked DNA)Nucleosomes (histones)30 nm chromatinLooped domains (non-histone protein scaffold)300 nm5. Condensed scaffolded looped domains (700nm)6. Metaphase chromosome (1,400 nm)

Histones– Positively charged proteins– first level of DNA packing– Leave DNA transiently during replication– Remain associated with DNA duringtranscription (change position and shape to allowRNA polymerase move along the DNA)

Packing is highlyspecific andprecise withparticular geneslocated in thesame places

Eukaryotic l/120080/bio31.swf

Control of Gene ExpressionNuclear level controls1) Chromatin remodeling “On/off” switch2) Transcription Factors3) Alternative splicing

Levels of control ofgene expressioninclude: Chromatin “remodeling”packing (Epigenetic) Transcription RNA processing:efficiency & alternativesplicing RNA stability-microRNAsTranslationProtein modificationsProtein degradation

Maximizing Genetic InformationFigure 11.11

Proteins discrete structuraland functional regionscalled domains. Different exonscode for differentdomains of aprotein.

Transcriptional Control1. Chromatin-modifying enzymes- availability of DNA for transcription (OnSwitch)2. Fine-tuning begins with the interaction oftranscription factors with DNA sequences

Chromatin Remodeling Long non-coding RNAs coating the chromatin Specific Chemical modifications that bind to histonesand DNA are:- Acetyl group- histones- Methyl groups- histones and DNA- Phosphate groups- histones

HistonetailsDNAdouble helixAmino acidsavailablefor chemicalmodificationHistone tails protrude outward from a nucleosomeUnacetylated histonesAcetylated histonesAcetylation of histone tails promotes loose chromatinstructure that permits transcription

Chromatin Chemical Modificationsa. DNA methylation Active genes- unmethylated (Euchromatin) Inactive genes- highly methylated (Heterochromatin)b. Histone acetylation Acetylated histones (Euchromatin) De-Acetylated histones (Heterochromatin)DNA methylation and histone de-acetylationcooperate to repress transcription

Transcriptional control Initiation of transcription- universal control ofgene expression Controlled by interaction between proteinsand DNA

Proximal and Distal Control elements ofgene transcriptionEnhancer(distal control elements)Proximalcontrol elementsExonIntronExonPoly-A signalTerminationsequenceregionIntron ExonDNAUpstreamDownstreamPromoterPrimary RNAtranscript 5′(pre-mRNA)TranscriptionExonIntron RNAIntronPoly-A signalExonIntron ExonCleaved 3′ endof primarytranscriptRNA processing:Cap and tail added;introns excised andexons spliced togetherCoding segmentmRNA3′Start Stop5′ Cap 5′ UTRcodon codon(untranslatedregion)3′ UTR Poly-A(untranslated tailregion)

Proximal controlelements- Control transcriptioninitiation- Binding sites forTATA binding proteinRNA polymeraseOther factors- Coordinated interactionof a massive assembly

Distal control elementsAction at a distance- Enhancers & Silencers (Tissue specific)- Located far from the promoter upstream,downstream, or within introns- Enhancers- bind activators- Silencers- bind repressorsTranscription and complex 437316/student g/a/index.html

Distal controlelementDNAActivatorsEnhancerBinding of an activator toenhancer sequencesbends DNA to makecontact with the proteininitiation complex at neraltranscriptionfactorsGroup ofmediator proteinsRNApolymerase IIRNApolymerase IITranscriptionInitiation complexRNA synthesis

Role of Activators in Differential Gene ExpressionLiver cellnucleusAvailableactivatorsLens ressedCrystallin genenot expressedLiver cellAlbumingene notexpressedCrystallin geneexpressedLens cell

Levels of control ofgene expressioninclude: Chromatin packingTranscriptionRNA processingTranslationVarious alterations tothe protein product Protein degradation

Post-transcriptional control mechanisms1. RNA processing2. Alternate splicing3. Half-life of RNA moleculepoly A tail5’ cap removalNucleotide sequences in the 3’untranslated (3’-UTR) trailer region4. RNA interference (non-coding RNA:miRNA, siRNAs and others)

Alternative RNA splicingRegulates coding sequence of mRNAExonsDNAPrimaryRNAtranscriptRNA splicingmRNAor

Non-coding interference RNAsSeveral groups:MicroRNAs (miRNA)- inhibit translation of same RNASmall interfering RNA (siRNA)- inhibit translation of other RNAsLong non-coding RNA (lncRNA)- variety of functions act incytoplasm and -rna/When a interfering RNA binds to a “target” mRNA, itprevents translation Specific degradation of an mRNA Specific blocking of translationThe human genome has about 1,000 distinct microRNAsthat regulate at least 1/3rd of the protein-encoding genes

Figure 11.7

RNA interference (RNAi)- Mechanism for silencing gene expressionthrough a technology that uses RNA moleculescalled RNA interference (RNAi)- Small synthetic, double-stranded RNAmolecules are introduced into selected cells toblock gene expressionProteincomplexDegradation of mRNADicerORmiRNATarget mRNAHydrogenbondBlockage of translation

Genome regulation by long cles/PMC3858397/

Translational control1. Protein factors required to initiate2. Specific sequences within the 5’-UTR(leader region) of mRNA bind toregulatory proteins preventing translation3. Interfering RNAs

Post-translational modificationsFunctional proteins- enzymatic cleavage- chemical modifications- transport to the appropriate destinationImproperly modified proteins are promptlydegraded

Normal proteins undergo selective degradation tolimit half-life marked by ubiquitin proteins (76 amino acids) Giant proteosomes recognize the ubiquitin anddegrade the tagged roteasomes.html

Piwi-interacting RNAs (piRNA)suppression of transposon activity ingermline and somatic a/

3. Identify the main mechanism for turning on gene expression. Explain why control of gene expression in eukaryotic cells is like a “dimmer switch”, an “ON” switch that can be fine tuned. 4. Identify the major switch and all the fine-tuning steps that can modulate eukaryotic gene expression. 5.