BIOL 4003 Week 10 Lecture Notes
BIOL 4003 Week 10 Lecture Notes 4003
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This 9 page Class Notes was uploaded by Rachel Heuer on Wednesday March 30, 2016. The Class Notes belongs to 4003 at University of Minnesota taught by Robert Brooker in Spring 2016. Since its upload, it has received 8 views. For similar materials see Principles of Genetics in Biology at University of Minnesota.
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Date Created: 03/30/16
Chapter 15: Eukaryotic Gene Regulation during Transcription - Eukaryotes derive many benefits from regulating genes o Respond to nutrient availability o Respond to environmental stress - Multicellularity and a much more complex cell structure also demand a much greater level of gene regulation - Gene regulation is necessary to ensure: o Genes are expressed in correct cell life cycle § Embryonic vs adult life stages o Genes are expressed in the correct cell type § Nerve vs. muscle cells o Fig 15.1 - Regulatory transcription factors: o Transcription factors are proteins that influence ability of RNA polymerase to transcribe a gene o 2 types: § General transcription factors: required for binding of RNA polymerase to core promoter and progression into the elongation phase • Necessary for basal transcription (low level of transcription) § Regulatory transcription factors: regulate rate of transcription of target genes • Influence ability of RNA polymerase to begin transcription of particular gene o 2-3% of human genes encode transcription o Activator (regulatory transcription factors) binds to the enhancer o Repressor (regulatory transcription factors) binds to silencer o Influence ability of RNA to recognize and bind to DNA § Can bind, but prevent elongation phase o Most regulatory transcription factors do not bind directly to RNA polymerase o Three common interactions that communicate the effects of regulatory transcription factors: § TFIID-direct or through coactivators § Mediator § Recruiting proteins that affect nucleosome composition o TFIID is the regulatory transcription factor that recognizes TATA box of core promoter § Activator protein helps binding of TFIID binding, or TFIID recruiting RNA polymerase § Repressor can prevent TFIID from binding à no RNA polymerase binding o Mediator § Wraps around RNA Polymerase § Activator can cause mediator to phosphorylate RNA polymerase, causing the initiation of elongation phase (TFIID is released) § Repressor does not cause mediator to phosphorylate RNA polymerase, so elongation phase does not start • Inhibits transcription o The function of regulatory transcription factors can be affected in three ways (Figure 15.6) § Binding of small effector molecule • Allows binding/not binding of transcription factor to DNA § Protein-protein interactions • Homo-dimer can bind to DNA but individuals cannot § Covalent modification - Chromatin remodeling and histones o ATP-dependent chromatin remodeling refers to dynamic changes in chromatin structure o These changes can range from a few nucleosomes to large scale changes § Carried out by diverse multiprotein machines that reposition and restructure nucleosomes o Chromatin structure: 3D packing is important factor for affecting gene expression § Chromatin is a very dynamic structure that switches between closed (not transcribable) and open (transcribable) conformations § Closed conformation • Chromatin is very tightly packed • Transcription may be difficult or impossible § Open conformation • Chromatin is not tightly packed • Chromatin is accessible to transcription factors • Transcription can take place o ATP-dependent chromatin remodeling § The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible § Alters gene expression o Chromatin remodeling can change relative position of nucleosomes § Can also cause different spacing between nucleosomes § Could remove whole histone or chromatin itself • Creates a gap with no nucleosome § Also could exchange a “variant” (nonstandard) histone for a standard one § Histone variants: • There are 5 histone genes that are moderately repetitive o H1, H2A, H2B, H3 and H4 • Human genome has over 70 histone genes o Most encode standard histones o A few of these genes have accumulated mutations that alters the amino acid sequence § These are termed histone variants § Creates specialized chromatin § Some histone variants enhance transcription (more easily removed) § Some histones decrease transcription (bind tightly to DNA) • Histone code: o Many enzymes can modify amino terminal tails of histones § Acetylation, methylation and phosphorylation are common § These modifications affect levels of transcription • May influence interactions between nucleosomes • Occurs in patterns recognized by proteins o Called the histone code o Pattern of modifications provide bindings sites for proteins that signal alterations which must be made to chromatin structure o Proteins bind based on the code and influence level of transcriptions § Acetylated can loosen DNA (easier to transcribe) - Chromatin Immunoprecipitation Sequencing has revealed a common pattern of nucleosome organization: o MAKE SURE YOU KNOW o Eukaryotic genes have a pattern, with nucleosome free regions at the beginning of a gene and at the end, which signals where transcription can occur o For transcriptional activation to occur, an activator can bind to an enhancer (in the nucleosome free region) § Can recruit a histone modifying or a chromatin remodeling complex • Makes is easier for preinitiation complex to form • Acetylated histones are easier to kick out (and put back on by chaperones) when strand is elongating § Hard for this to do if histones are in a closed conformation - DNA Methylation: o DNA methylation is a change in chromatin structure that usually silences gene expression o Carried out by an enzyme called DNA methyltransferase § Recognizes a CG sequence in one strand and a GC sequence in the opposite strand o Common in some eukaryotic specieis, but not all § Level of methylation varies among eukaryotes o Can be unmethylated, hemimethylated, and fully methylated. § Need two cytosines kitty corner from one another (on opposite strands but one nucleotide away from eachother) • Only the occurs on cytosine § Hemimethylated: only one of the cytosines is methylated § Fully methylated: both cytosines are methylated § Unmethylated: none of the cytosines are methylated o DNA methylation usually inhibits the transcription of eukaryotic genes § especially when in the vicinity of the promoter o Plants and vertebrates contain CpG islands near their promoters § Islands are typically 1,000-2,000 nucleotides long, with many C-G bonds (which can be methylated) o In housekeeping genes (where gene is expressed in most cell types), CpG islands are unmethylated § Because these genes are expressed in most cell types o In tissue specific genes, genes are often silenced by methylation of CpG § Methylation may influence binding of transcription factors § Can recruit methyl-CpG-proteins that can compact the chromatin o CpG methylation can restrict activator from binding to DNA o Could allow CpG binding protein which recruits other repressors Chapter 16: Translational Regulation in eukaryotes - Epigenetics: The study of mechanisms that lead to changes in gene expression that can be passed from cell to cell and are reversible, but do not involve a change in the sequence of DNA o Epigenetic inheritance involves epigenetic changes that are passed from parent to offspring § Such as genomic imprinting o Different molecular changes underlie epigenetic regulation § DNA methylation § Chromatin remodeling § Covalent histone modification § Localization of histone variants § Feedback loops o How does epigenetics/gene targeting start? § Some epigenetic changes are initiated by transcription factors • Transcription factors binding causes change in structure of strand (like methylation) § Non-coding RNA can recognize specific gene regions and recruit proteins that modify the gene o Epigenetic changes can be Cis- or trans- § Cis-epigenetic changes are maintained at a specific site • Only one copy of a gene § Trans-epigenetic changes are maintained by diffusible factors, such as transcription factors • Affects both copies of a gene o Two General Categories of epigenetic gene regulation § Epigenetic gene regulation may occur as a programmed developmental change or be cased by environmental agents o Can be programmed during development § Can be programmed by developmental change or by environmental agents • Temperature, diet, toxins o Development: series of genetically programmed stages in which a fertilized egg becomes an embryo and eventually an adult § Many changes that occur during development are maintained by epigenetic regulation § Three examples: • Genomic imprinting • X-chromosome inactivation • Formation of specific cell types and tissues § Genomic imprinting • Gene flanked by an ICR (imprinting control region) and a DMR (differentially methylated region) o these regions don’t get methylated in females, and CTC factors bind to each region and link them together to create a loop o Loop prevents enhancer from turning on the IGF2 gene o This is maintained in offspring and cell division • In spermatogenesis, ICR and DMR become methylated o This prevents the CTC factor from binding so there is no loop formation o The enhancer can turn on the expression of the IGF2 gene § X-chromosome inactivation: • X-chromosome inactivation (XCI) occurs during embryogenesis in female mammals • A portion of the X chromosome called the X inactivation center (XIC) plays a key role o Prior to XCI, the Tsix gene is expressed on both X chromosomes • The XIC encodes two genes, Xist and Tsix, which are transcribed in opposite directions • Before x-inactivation, pluripotency factors bind to Tsix gene, stimulating Tsix transcription on both strands o This leads to X-chromosome pairing (due to binding of CTCF factors) prior to x- chromosome inactivation • To inactivate, chromosome pairs break, and one x- chromosome gets all the pluripotent factors o X-chromosome that gets pluripotent factors remains active • Chromosome without pluripotent factors can now transcribe Xist gene o Transcription factors bind Xist RNA to the chromosome o Xist RNA continues to be transcribed repeatedly. o Another protein keeps tethering new Xist RNA to the chromosome (throughout the spreading phase) o Other stuff gets recruited to inactive X- chromosome to create a Barr body (compacted) • Tsix must only be transcribed once in the whole process o Epigenetic changes occur during embryonic development that are remembered throughout subsequent cell divisions § Muscle cells have nerve cell genes repressed § Some genes are permanently “turned off” in certain cell types while others are not § Two types of competing protein complexes are key regulators of epigenetic changes during development that produce specific cell types and tissues • Trithorax group (TrxG)– involved with gene activation • Polycomb group (PcG) – involved with gene repression o 2 complexes (PRC1 and PRC2) o First PRE binds to DNA and recruits PRC2 o PRC2 recruits methyl groups to lysine 27 of histone tails § Can inhibit RNA polymerase binding § Could recruit PRC1 to target gene § PRC2 may not be needed for PRC1 activity o PRC1 can interact with TFIID inhibiting its ability to recruit RNA polymerase to the promoter (or from preceding to elongation phase) § PRC1 can also cause chromatin compaction or the covalent modification of histones o Environmental agents and epigenetics § Many environmental agents have been shown to cause epigenetic changes § These include dietary effects as well as toxins in the environment § Agouti gene in mice promotes synthesis of yellow fur pigment • In a strain of mice, a transposable element carrying a promoter is inserted upstream from the Agouti gene; this is called the A allele o Gives gene two promotes o Stronger promoter (in element) leads to more production of yellow fur o Range of color intensity despite same genotype § Strains of mice carrying the A allele show a range of coat colors, from yellow to pseudo-agouti o When diet contained chemicals that lead to increases in DNA methylation, offspring had darker fur § Consistent with idea that DNA methylation inhibits the agouti gene from being transcribed § Cancer: • Methylation, chromatin remodeling, and histone modification can turn off suppressor genes that inhibit cancerous growth - Regulation of RNA processing and translation o Eukaryotes use this method much more than bacteria o Alternative splicing: pre-mRNA can be spliced in more than one way § Can create different polypeptides § In most cases, large sections of the coding regions of proteins are often the same, resulting in alternative versions of a protein that have similar functions • Each polypeptide still has its own characteristics § Constitutive exons are in all versions of mRNA translation § Alternative exons can be included or excluded • Often cell-specific § Alternative splicing allows increasing specificity § Splicing factors recognize parts of pre-mRNA, can bind to 3’ end of repressor, causing 3’ end of an exon to be ignored and more gene to be spliced out • Known as exon skipping § Some splice sites are not recognized well • Splicing enhancers can increase the ability of the spliceosome to recognize the splice sites § Each cell type has specific combinations of splice repressors and splice enhancers o Stability of eukaryotic mRNA varies greatly § Minutes to months § Stability can be regulated so that its half-life is shortened or lengthened • This influences mRNA concentration and gene expression § Factors that can affect mRNA stability • Poly A tail length • Destabilizing elements § Double stranded RNA can silence expression of certain genes • RNA interference is mediated by microRNAs • microRNAs (miRNAs) or short-interfering RNAs (siRNAs) cause RNA interference • miRNAs are encoded by genes in eukaryotic organisms o do not encode a protein o give rise to small RNA molecules o Do not perfectly match to mRNAs that they bind to • siRNAs form from two RNA molecules that form a double stranded region o usually close to a perfect match to mRNAs • microRNA gene is transcribed into a primary mRNA (pri-mRNA) • folds into stem loop, which is exported out of the nucleus (becomes pre-mRNA) o siRNA can just be imported right away • Double stranded region is clipped by dicer protein, one strand forms RNA inducing silencing complex (RISC) • RISC binds to specific messenger RNA • Figure 16.12 (called RNA interference) • If miRNA that’s not a perfect match, it can be exported to pbody where it can be stored or degraded • siRNA will match mRNA and will be degraded • called silencing because it is either sent to a p-body, where it is not translated, or it is degraded o known as RNA interference • Benefits of RNA interference: o New form of regulation o Defense mechanism against viruses § RNA viruses that have/produce double- stranded RNA in their genome/reproductive cycle o Can silence transposable elements § Iron assimilation and translation • Regulation during translation • Iron is needed for the function of many enzymes, but is toxic in wrong concentration • Iron is taken up by transferrin protein o Iron will be used as a cofactor or stored in ferritin if there is excess (iron bubbles) • Translation of ferritin mRNA is inhibited when iron levels are low due to the binding of IRP (iron regulatory protein) o Repressor binds to mRNA, preventing translation • Iron can be cofactor for IRP, allowing transcription to occur • At high iron levels, iron binds to IRP, which is released from the IRE o Translation can occur § When iron levels are low, IRE binds to the 3’ end of mRNA [increases stability] • More stable = more translation § High iron levels = no binding of IRP = low stability = degradation = no translation