(Ill) A rock is dropped from a sea cliff and the sound of itstriking the ocean is heard 3.4 s later. If the speed of soundis 340 m/s, how high is the cliff?
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