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Gene Regulation in Eukaryotes

    Medium, 8 examples, 5 practice questions

  • Eukaryotic gene regulation is much more complicated than in prokaryotes, utilizing more regulators and larger regulatory sequences.
  • Signal transduction allows extracellular molecules to cause a chain of events that causes a biochemical response inside the cell.
  • The RNA-induced silencing complex (RISC) is a ribonucleoprotein complex whose most well studied function is the degradation of target mRNA, which decreases the level of transcripts available to be translated.
  • Translational regulation can be specific or global.
  • Epigenetics concerns the inheritance of patterns of gene regulation not found in the DNA sequence itself.

Gene Regulation in Eukaryotes

What is the name of the process where an extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell?
  • Transcriptional regulation
  • Translation-dependent regulation
  • Signal transduction
  • Epigenetics
Proteins found within cells that are responsible for sensing steroid and thyroid hormones and certain other molecules are called:
  • Ligands
  • Nuclear receptor proteins
  • Polymerases
  • Hormones
What process is very important for gene silencing and viral infection defense?
  • JAK/STAT pathway
  • RNA interference
  • Signal transduction
  • Epigenetics
A type of translation-dependent regulation that rescues ribosomes that are translating mRNAs lacking a STOP codon is called:
  • Nonsense-mediated mRNA decay
  • Nonstop-mediated mRNA decay
  • Signal transduction
  • RNA interference
Methylation of DNA and nucleosomes being passed from generation to generation is an example of:
  • Genetics
  • Molecular Biology
  • Epigenetics
  • DNA replication

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.


Gene Regulation in Eukaryotes

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  1. Intro
    • Lesson Overview
      • Eukaryotic Transcriptional Regulations
      • Example 1
        • Locus Control Regions
        • (Ligand) Signal Transduction
        • N F Kappa B
        • Example 2
          • JAK/ STAT Pathway
          • Example 3
            • Seven-Spanner Receptors
            • Example 4
              • Nuclear Receptor Proteins (NRPs)
              • RNA Interference
              • RISC Pathway
              • Translational Regulation
              • Translation-Dependent Regulation
              • Epigenetics
              • DNA Methylation
              • DNA Methylation
              • Example 5
                • Nucleosomes
                • Chromosome Condensation Via J1
                • Histone Code
                • Example 6
                  • Nucleosome Assembly
                  • Example 7
                    • Chromatin Remodeling
                      • Example 8
                      • Polycomb Repressors
                      • Intro 0:00
                      • Lesson Overview 0:06
                      • Eukaryotic Transcriptional Regulations 0:18
                        • Transcription Factors
                        • Insulator Protein
                      • Example 1 1:44
                      • Locus Control Regions 4:00
                        • Illustration
                        • Long Range Regulatory Elements That Enhance Expressions of Linked Genes
                        • Allows Order Transcription of Downstream Genes
                      • (Ligand) Signal Transduction 8:12
                        • Occurs When an Extracellular Signaling Molecule Activates a Specific Receptor Located on the Cell
                        • Examples
                      • N F Kappa B 10:01
                        • Dimeric Protein That Controls Transcription
                        • Ligands
                      • Example 2 11:04
                      • JAK/ STAT Pathway 13:19
                        • Turned on by a Cytokine
                        • What is JAK
                        • What is STAT
                        • Illustration
                      • Example 3 17:00
                      • Seven-Spanner Receptors 20:49
                        • Illustration: What Is It
                        • Ligand Binding That Is Activating a Process
                        • How This Happens
                      • Example 4 24:23
                      • Nuclear Receptor Proteins (NRPs) 28:45
                        • Sense Steroid and Thyroid Hormones
                        • Steroid Hormones Bind Cytoplasmic NRP Homodimer
                        • Hormone Binds NRP Heterodimers Already Present in the Nucleus
                        • Unbound Heterodimeric NRPs Can Cause Deacetylation of Lysines of Histone Tails
                      • RNA Interference 32:01
                        • RNA Induced Silencing Complex (RISC)
                        • RNAi
                      • RISC Pathway 34:34
                        • Activated RISC Complex
                        • Process
                        • Example
                      • Translational Regulation 41:17
                        • Global Regulation
                        • Competitive Binding of 5 Prime CAP of mRNA
                      • Translation-Dependent Regulation 44:56
                        • Nonsense Mediated mRNA Decay
                        • Nonstop Mediated mRNA Decay
                      • Epigenetics 48:53
                        • Inherited Patterns of Gene Expression Resulting from Chromatin Alteration
                        • Three Ways to Happen
                        • DNA Sequence Does Not Act Alone in Passing Genetic Information to Future Generations
                      • DNA Methylation 50:57
                        • Occurs at CpG Sites Via DNA Methyltransferase Enzymes
                        • CpG Islands Are Regions with a High Frequency of CpG Sites
                        • Methylation of Multiple CpG Sites Silence Nearby Gene Transcription
                      • DNA Methylation 53:46
                        • Pattern Can Be Passed to Daughter Cells
                        • Prevents SP1 Transcription Factors From Binding to CpG Island
                        • MECP2
                      • Example 5 55:27
                      • Nucleosomes 56:48
                        • Histone Core
                        • Histone Protein
                      • Chromosome Condensation Via J1 57:32
                        • Linker Histone H1
                        • Compact DNA
                      • Histone Code 57:54
                        • Post-translational Modifications of N-Terminal Histone Tails is Part of the Epigenetic Code
                        • Phosphorylation, Acetylation, Methylation, Ubiquitination
                      • Example 6 58:52
                      • Nucleosome Assembly 59:13
                        • Duplication of DNA Requires Duplication of Histones by New Protein Synthesis
                        • Old Histones are Recycled
                        • Parental H3-H4 Tetramers
                      • Example 7 1:00:05
                      • Chromatin Remodeling 1:01:48
                      • Example 8 1:02:36
                        • Transcriptionally Repressed State
                        • Acetylation of Histones
                      • Polycomb Repressors 1:03:19
                        • PRC2 Protein Complex
                        • PRC1 Protein Complex
                        • MLL Protein Complex

                      Transcription: Gene Regulation in Eukaryotes

                      Hello, and welcome back to

                      Today's lesson will be on gene regulation in eukaryotes.0003

                      First, we will talk about transcriptional regulation and we will also talk about the regulation of translation.0008

                      Finally, we will talk just a little bit on epigenetics.0013

                      Let us first talk about our eukaryotic transcriptional regulators.0020

                      Mainly, we are going to talk about our transcription factors.0025

                      There are many different types of transcription factors, usually characterized by the specific domain by which they bind DNA.0028

                      We have homeodomain proteins, we have zinc finger proteins, we have leucine zippers, loop helix proteins, as well as HMG proteins.0037

                      HMG just stands for high mobility group.0052

                      We also have insulator proteins, in which these are proteins that bind between the enhancer and the promoter in a gene.0055

                      They suppress transcription.0065

                      If you remember, enhancers are usually far upstream of where the core gene element itself starts.0066

                      Up here, our transcription factors, they can be either enhancing or repressive.0076

                      Compared with our prokaryotes, transcriptional regulation and regulation in general, for eukaryotes is much more complex.0083

                      Usually, there are more regulators and longer length of regulatory sequence in the DNA.0094

                      Let us first start off with an example of transcriptional regulation.0106

                      We have a gal-4 and gal-1.0110

                      Gal-4 is a protein and gal-1 is a DNA sequence.0113

                      We are looking at this in the yeast saccharomyces cerevisiae.0118

                      The gal-4 protein will bind to a site upstream of the gal-1 gene.0125

                      This is going to increase transcription by gal-1 gene, by a thousand fold.0138

                      What this would look like, gal-4 binds as a dimer.0145

                      What we see here, if we draw this out.0150

                      Right here, this right here, this arrow, that shorthand form of saying that is where promoter is and gal-1 that is our gene.0170

                      This right here is a UAS, when upstream activating sequence.0179

                      There are 4 sites here at which the gal-4 protein will bind.0186

                      It will bind as a dimer.0198

                      Between the UAS and the gal-1 gene, is about 235 base pairs.0200

                      The binding upstream over here will affect how this gene is transcribed.0213

                      We can increase, this is gal-4, there are g4.0221

                      This can cause an increase of transcription about a thousand fold.0226

                      For example you are supposed to only make one mRNA0231

                      or in a certain period of time you make a thousand times more than that.0235

                      Let us talk about our locus control regions.0242

                      First off, we are going to draw this out.0245

                      I guess we will use red.0251

                      Here is our locus control region.0256

                      Then, we have our globin genes.0267

                      That should be gamma not Δ.0294

                      This is Δ, this one is β.0307

                      Here is our cluster of globin genes.0320

                      We have locus control regions right.0340

                      Locus control regions are just long range regulatory elements that enhance expression of our linked genes.0342

                      This helps function in a copy number dependent manner.0353

                      It is actually tissue specific.0360

                      For this one, for example, this allows this locus control region.0362

                      This allows ordered transcription of downstream genes.0369

                      What that is really saying is that we have selective expressions of our β globin genes in our erithrocyte cells.0375

                      This is an example talking about our selective.0406

                      Our erithrocyte cells are also known as red blood cells.0413

                      Our locus control regions allow for order transcription in downstream genes.0416

                      Here is our locus control region.0421

                      Here are downstream genes.0425

                      We have our 5 globin genes.0427

                      We have ε, we have gamma G, gamma A, Δ and β.0429

                      During human development, all 5 genes are transcribed sequentially, meaning, first ε, all way through to β.0433

                      What happens here is that we have euchromatin position.0442

                      If we remember correctly, euchromatin is open, more transcriptionally available.0447

                      Heterochromatin is closed, less transcriptionally available.0452

                      Euchromatin position will change and heterochromatin formation, reformation will follow behind.0456

                      This is something that I want you to just keep in mind because we will be talking about epigenetics later.0463

                      How euchromatin and heterochromatin positioning affects the transcription of genes,0472

                      as well as the replication of DNA sequence.0480

                      Here are locus control regions, this is one example.0488

                      Let us move on to signal transduction.0494

                      Signal transduction occurs when we have an extracellular signaling molecule,0498

                      activating a specific receptor located on the membrane of the cell.0506

                      If I draw it out, here is a cell and here might be a receptor.0514

                      Here is our extra cellular signaling molecule.0524

                      When this molecule binds the receptor, it is going to trigger a chain of events, inside the cell it creates some sort of response.0529

                      That response is going to be different, based on which pathway is being activated.0544

                      Some of our examples that we will talk about today are NF kappa B,0550

                      the JAK/ STAT pathway which is very important for immune system regulation.0555

                      We have sigma molecule growth factors, seven-spanner receptors which are important for sensing light, smells, serotonin.0559

                      And then, we have nuclear receptor proteins that are important for the signaling of our steroid and non-steroid molecules.0572

                      This is an important one that you will learn more in biochemistry.0590

                      If you have not taken that yet or if you decide to take a nutrition class, something like that.0594

                      First off, NF kappa B.0603

                      NF kappa B is a dimer protein meaning it has two parts.0607

                      We have to have two pieces of that protein.0612

                      This protein helps control transcription, as well as cytochime production in cell survival.0617

                      Cytochimes are just small protein molecules that are involved in immune system regulation.0622

                      Ligands which means it is just something that is binding, can cause our NF kappa B0628

                      to turn on more than 150 genes in the inflammatory system and the immune system,0637

                      as well as during development in the cytoplasm.0642

                      I mentioned that the NF kappa B is a dimer.0646

                      That dimer is made up of the P50 and the P60 protein, therefore, it is a heterodimer.0650

                      This might be 50, this would be 65, then we have our heterodimer.0657

                      Let us talk a little bit more about NF kappa B.0662

                      NF kappa B, as I said, plays a large part in the immune system and so forth.0667

                      NF kappa B, for one example, can be inhibited when the P50 subunit binds to a cancer suppressor protein.0672

                      If it is inhibited, therefore, it cannot help with transcription or increase transcription, for the rest of its purposes.0686

                      A second example of how NF kappa B maybe regulated, it can be inhibited by its binding to another protein called I kappa B.0696

                      NF kappa B is bound or it can be bound and inhibited by the inhibitory protein I kappa B.0710

                      What you can do to release this inhibition is phosphorylate I kappa B, that will cause I kappa B to release NF kappa B.0720

                      At this point, NF kappa B can travel to the nucleus, it was originally in the cytoplasm.0734

                      At that point, once NF kappa B is in the nucleus, it can go ahead and regulate any transcription, as it normally would.0743

                      At the same time, this phosphorylated I kappa B is no longer bound to NF kappa B, can be ubiquitinated.0752

                      Ubiquity, if we remember back to the protein unit, is just a small protein and that can have many different outcomes.0761

                      In certain outcomes, when a protein is ubiquitinated, it is set to the prodiuzome and degraded,0771

                      broken down, thrown into the trash.0778

                      These are two separate examples how NF kappa B can be regulated, in which case,0781

                      if it is inhibited, it cannot affect transcription.0785

                      If it is not inhibited and able to act in the nucleus, then it can affect and be a regulatory protein.0791

                      Next, we have the JAK/ STAT pathway.0801

                      This pathway is turned on by a cytochime.0803

                      For an example, interferon α, that will attach to the cell surface receptors.0807

                      First of all, let us write out what is JAK.0812

                      JAK is a kinase, it is called Janus kinase.0823

                      Remember, kinases are responsible for phosphorylating things.0832

                      We have STAT, STAT stands for signal transducer and activator of transcription.0837

                      The JAK/ STAT pathway, if we are talking about the previous slide.0878

                      If we have our cell, here is our nucleus.0887

                      We have a receptor and we have something binding to that receptor.0893

                      In this case, the cytokine is binding.0902

                      JAKs, the kinases come together.0906

                      Those JAKs will activate the kinase which phosporylates pairs of our STAT.0911

                      Those phosphorylated STAT monomers will dimerize.0919

                      The stats are also monomers.0923

                      They will be phosphorylated.0933

                      We will just say that it is a yellow piece, that causes them to dimerize.0934

                      They do that via a specific domain on the STAT molecule called an SH2 domain.0954

                      We will call the nucleus in purple over here.0970

                      That STAT dimer will travel into the nucleus and bind a specific DNA sequence, related to transcriptional enhancement.0975

                      This, once again, shows you that a molecule from outside the cell can affect what happens inside the cell,0987

                      through a series of events that happens to, in this case, deal with the binding of a molecule to receptor,0995

                      a phosphorylation of other proteins.1006

                      Those proteins being sent into the DNA or being sent into the nucleus, to bind to DNA.1009

                      At that point, it will affect transcription.1016

                      Another example of sigma transduction would be our signal molecule growth factor.1022

                      A couple examples being epidermal growth factor or EGF, as well as insulin.1028

                      Remember, insulin is the protein that is sent out by the body, by the pancreas, in response to glucose in the blood, blood sugar.1033

                      When you eat something, you have carbs, those get broken down, absorbed from the stomach through the intestine into the blood.1046

                      Insulin is released, insulin takes that glucose into the cells.1055

                      How this works is that, we have a binding to the receptor, activating a dimeric, a two protein,1061

                      RTK which is a receptor thyrosine kinase enzyme.1076

                      The dimerization requires the adapter proteins grab to an SOS.1082

                      That is just some extra information for your knowledge.1088

                      We have this signal causing the RTK dimerization.1092

                      That dimer binds and activates a protein called RAS, which will bind the GTP molecule.1102

                      That RAS will then bind to and activate another protein called RAF.1110

                      RAF is what is called a map KKK which is monogen activated protein, we usually call this MAP.1115

                      It is a MAP kinase kinase kinase meaning it phosphorylates a MAP kinase kinase.1127

                      It is quite heavy nomenclature but we will understand it, as we go to the next step.1138

                      This RAF which is our enzyme, a specific MAP kinase kinase kinase.1143

                      RAF will then phosphorylate, since it is a kinase, it will phosphorylate MEK.1149

                      MEK is a MAP kinase kinase.1155

                      MEK, once it is phosphorylated, will phosphorylate a MAP kinase, a MAP K.1162

                      This MAP K is a different type of kinase, than the one that we talked about up here,1175

                      it is a kinase that is called a serine threonine kinase.1182

                      What are the differences between these kinases?1187

                      The RTKs, they phosphorylate a thyrosine amino acid.1189

                      This one down here will phosphorylate proteins at certain amino acids of either a serine or a threonine residue.1199

                      Now that MAP kinase is active, it is still a kinase.1213

                      MAP kinase will phosphorylate a bunch of other transcription factors.1218

                      These transcription factors, now many of them by the phosphorylate, will be activated.1224

                      And now, they can go and bind to the DNA in the nucleus and enhance transcription.1231

                      It is a very complex process, many steps, but the whole outcome is to affect the transcription happening in the nucleus.1239

                      We have other ways of regulating transcription.1252

                      Utilizing certain proteins called seven-spanner receptors.1257

                      What is a seven-spanner receptor?1262

                      If this is the cell membrane, we have a protein that crosses the cell membrane 7 times, 1, 2, 3, 4, 5, 6, 7.1266

                      Maybe that is its N terminal tail and this is its C terminal tail.1284

                      What we have here is it is crossed 7 times, 1, 2, 3, 4, 5, 6, 7.1288

                      That is what is a seven-spanner receptor is.1296

                      There are several different seven-spanner receptors.1299

                      How do these work?1303

                      Very similar, in the fact that there is ligand binding that is activating a process.1306

                      Specifically, we have ligand binding, activating trimeric proteins called G proteins.1313

                      Trimeric meaning 3, 3 different polypeptide chains.1320

                      Each one of these subunits, we have an α subunit, a β subunit, and a gamma subunit.1328

                      In order, how this happens, we have the ligand binding activating that protein.1339

                      When GTP binds, our α subunit that will activate adenylyl cyclase, which is an enzyme.1347

                      This requires GTPAs activating protein also known as GAP.1363

                      Adenylyl cyclase normally converts ATP to cyclic AMP.1372

                      We talked about this in the previous lesson, this adenylyl cyclase enzyme.1376

                      The CAMP, we have also talked about this, will bind to protein kinase A to activate it.1383

                      Protein kinase A, otherwise known as PKA, will then be able to phosphorylate many different proteins.1391

                      Some of which can be transcription factor.1401

                      PKA, once activated can phosphorylate transcription factors which will bind to certain pieces of DNA in the nucleus and affect transcription.1403

                      Protein kinase A can also another protein called CREB which will bind to a specific DNA sequence called CRE.1416

                      As well as, be bound by CBP and P300 protein which is CBP stands for creb binding protein, CREB BP.1429

                      CBP300, this protein is normally considered a transcriptional activator.1444

                      It turns on the transcription of genes.1450

                      There are two different ways a protein kinase can affect gene expression.1453

                      Let us talk about another example.1465

                      We actually have two examples in this one.1467

                      Let us talk about how we have two different diseases, cholera and whooping cough, otherwise known as pertussis.1470

                      Cholera, cholera is caused by the action of the bacteria vibrio cholerae.1479

                      This vibrio cholerae produces a toxin called the cholera toxin.1495

                      How does this work, what is the mechanism of action?1500

                      The cholera toxin catalyzes an ADP ribosylation.1504

                      That is basically adding a ribose group to ADP together.1509

                      This happens via the use of our old friend NAD, nicotinamide adenine dinucleotide.1514

                      We have talked about this one before and it is useful in energy.1523

                      This ATP ribose will bind to the α subunit of the G protein, as we talked about in previous slide,1529

                      which does not allow the GDPAs activating protein, GAP, to activate GTPAs.1538

                      Therefore, you cannot convert GTP to GDP.1545

                      What is that even mean?1551

                      What that means that we have constitutive activation of our adenylyl cyclase enzyme.1553

                      Constitutive means it is always on.1562

                      If our adenylyl cyclase enzyme is always on, that means that we are continually turning ATP into CAMP.1566

                      If we have a bunch of CAMP, that is going to keep PKA, protein kinase A, constitutively active.1577

                      If PKA is always active, it is always going to be phosphorylating things.1587

                      In these certain cases, it will phosphorylate certain transcription factors to turn on gene expression and1594

                      can lead to types of diseases or the types of symptoms from cholera or whooping cough that are common.1601

                      Whooping cough, specifically, is caused by the bacteria bordetella pertussis and1614

                      that bacteria also produces a toxin called the pertussis toxin.1621

                      This pertussis toxin acts in a little different way than the cholera toxin does.1625

                      Let us talk about this, we have pertussis toxin binding to a protein called G sub I.1632

                      G sub I is a G protein inhibitor.1640

                      Normally, G sub I will bind to and inhibit adenylyl cyclase, opposite of cholera.1647

                      Cholera adenylyl cyclase is always on.1660

                      G sub I normally turn adenylyl cyclase off.1663

                      However, we have pertussis toxin binding to that inhibitor preventing that normal inhibition.1667

                      Therefore, we end up with the same outcome.1680

                      We have constitutive activation of adenylyl cyclase, leading to continual conversion of ATP to cyclic AMP.1683

                      Meaning, continual activation or constitutive activation of protein kinase A,1694

                      leading to transcriptional regulation at increased rates.1701

                      These are just a couple of ways how we can utilize the previous material in this unit,1708

                      to understand how a couple different bacteria create their symptoms that we see for medical purposes.1715

                      Going on to a different type of transcriptional regulation.1728

                      We have what are called nuclear receptor proteins.1732

                      These are proteins found within cells that are responsible1736

                      for sensing both steroid and thyroid hormones, as well as certain other molecules.1739

                      First off, we have our steroid hormones.1749

                      For example, our androgens and estrogens, our sex hormones, our glucocorticoids which are involved in water retention.1752

                      They are going to bind our cytoplasmic nuclear receptor protein homodimer.1761

                      Let us say dimer of the same two things, two different polypeptides.1768

                      The steroid hormones, these will bind our NRP homodimer.1774

                      This is what is called a type 1 complex.1782

                      The type 1 complex will head the nucleus, bind to an inverted repeat DNA sequence called a hormone response element or an HRE.1787

                      At that point, once the HRE is bound then transcription can be regulated, either in a positive or negative manner.1802

                      A second way that nuclear receptor proteins act is sensing hormones, such as vitamin A or a thyroid hormone.1811

                      They will bind to NRP heterodimers.1822

                      In this case, it is two different proteins.1826

                      This is what is called a type 2 complex.1836

                      This type 2 complex is already in the nucleus.1840

                      It will bind direct repeats in the nuclear DNA and then affect transcription regulation.1845

                      A third way to look at our NRPS and how they might affect transcription1855

                      would be in an unbound heterodimic NRP, not bound by hormone.1862

                      They can cause deacetylation of histone tails, the lysine residues on histone tails.1868

                      These heterodimeric NRPs that are bound by a hormone, such as this right here, can cause the acetylation of those tails.1876

                      Remember, when a histone, when its tails are acetylated, depending on the histone code, it is very complex.1887

                      It often is related to the loosening of chromatin meaning transcriptional activation.1899

                      Whereas, the deacetylation of histone tails usually means the compaction of chromatin,1908

                      meaning decreased transcription and replication.1915

                      Let us move on to something that is also regulating transcription but in a completely different way.1923

                      Actually, this is a really cool mechanism.1932

                      This is what is called RNA interference.1937

                      This is a method that is actually been adopted by many research labs.1939

                      I will show you an example in a couple slides to actually choose what genes get silenced or expressed.1945

                      This is coordinated by a complex called the RISC complex.1958

                      The RNA induced silencing complex.1964

                      It is a ribonuclear protein complex meaning it has both RNA and protein.1970

                      This complex has been most well studied for having a function in the degradation of a specific target mRNA.1977

                      If you target mRNA to be degraded, that will decrease the level of the transcripts available to be translated.1988

                      Therefore, in S sense it is functioning to decrease gene expression of a specific gene.1996

                      We have very important players.2006

                      We have RNase enzymes, RNase is an enzyme that breaks down RNA molecules.2009

                      These RNase enzymes called dicer and drosha, trim double stranded RNA2016

                      to form either small interfering RNA, sRNA, or microRNA, miRNA.2024

                      There RNAs then get incorporated into the RISC complex leading to specific mRNA targeting, and then degradation.2035

                      It targets to a specific mRNA and does not allow translation to take place.2046

                      It is actually really cool.2052

                      This process we, call RNA interference or RNAi.2055

                      We find this in many eukaryotes.2061

                      It was thought to be developed as a very important mechanism of gene silencing and viral infection defense.2063

                      Let us talk about the RISC pathway, in a little bit of detail.2076

                      First of all, what is an activated RISC complex?2080

                      That composes the RISC ribonuclear protein, as well as a micro RNA or small interfering RNA and an ATP molecule.2083

                      How does this all go down?2096

                      First off, dicer and drosha cleave a double stranded RNA into short 21 to 23 base pair fragments,2100

                      with a two nucleotide 3 prime overhang.2111

                      Simply, it looks like this, where this is two nucleotides, that is two nucleotides long.2115

                      This is our 3 prime, 5 prime, 3 prime, 5 prime.2137

                      This whole thing is 21 to 23 base pairs.2145

                      This is double stranded RNA.2156

                      That is the first thing that happens.2165

                      Then, we bring in a different RNase called argonaut which is also termed slicer.2167

                      The RNase argonaut then associates the single stranded RNA with the RISC complex,2178

                      to act as a complementary strand to whatever the target mRNA is.2190

                      This double stranded piece right here, that has been processed by dicer and drosha2198

                      can be separated into single strands and that is what argonaut is working with.2206

                      The complex will bind to our target mRNA, whatever piece of gene material2210

                      that you do not want to end up turning into a protein.2219

                      The complex binds to the target mRNA and silences it.2223

                      Silencing meaning it is not been turned into a protein, no translation.2226

                      There are two different ways this occurs.2232

                      If we are talking about an miRNA being used, micro RNA.2234

                      The micro RNA in the RISC complex binds to the 3 prime untranslated region,2242

                      that is a part of an RNA, and is untranslated.2249

                      Therefore, it would not have turned into protein anyway2252

                      but is a great spot for regulatory proteins to bind and affect whether something is translated or not.2256

                      MiRNA in that RISC complex, binds to the 3 prime UTR with a mismatch.2262

                      It will block transcription.2270

                      The other way that we can affect translation, if we have a small interfering RNA in the RISC complex,2274

                      binding to the mRNA without a mismatch.2285

                      This will cause RISC to cleave the mRNA.2291

                      Cleaving it vs. just blocking transcription.2296

                      mRNA degradation happens at a certain spot in the cell called a P body.2304

                      mRNA degradations localized in P bodies which are found in cytoplasmic space.2314

                      And often stain darkly, that you can look under a microscope.2321

                      Importantly, we have activated RISC, the RNA inducing complex,2329

                      being implicated in formation of nuclear heterochromatin.2339

                      Meaning, we can have RISC actually not just cleaving these mRNAs but can actually somehow be a part of epigenetic changes.2342

                      Actually not even getting transcription available, not even allowing transcription to happen.2358

                      That is kind of cool.2364

                      Here is an example of the RISC pathway.2369

                      Actually, how scientists have been able to utilize it.2371

                      What we would have is viral, if there is an infection, they make double stranded RNA.2378

                      You can break that into srRNA via dicer and drosha.2386

                      Those double stranded RNAs can enter into the RISC complex, argonaut can take a single stranded RNA2391

                      and utilize that either as an miRNA or srRNA, to either degrade the mRNA2398

                      or to affect possibly the epigenetic state of the heterochromatin.2406

                      What scientists can do, they can target individual genes and silence them.2414

                      Here we see a wild type, a normal petunia plant.2420

                      These two are lab created petunias, utilizing RNA interference, RNAi, using the RISC pathway.2426

                      What they have done is through selective targeting of the mRNA specific for the color,2442

                      they have silenced them in the white portions.2448

                      This is pretty cool.2453

                      RNAi can be used other than just for cool phenotypes.2455

                      It can actually possibly be used for more functional things such as something that we will talk about a little later,2464

                      maybe gene therapies, something similar to that.2471

                      That is your transcriptional regulation.2479

                      We will just have a few slides, a few instances of translational regulation.2482

                      Just like prokaryotes, eukaryotes mostly regulate transcriptionally.2486

                      However, they can regulate translationally.2494

                      You can have global regulation of translation meaning you regulate all translation,2498

                      not specific genes, by phosphorylating protein EIF2 that we talked about before.2505

                      This is eukaryotic initiation factor 2.2515

                      If you phosphorylate EIF2 that protein cannot bind GTP.2519

                      If you cannot bind GTP, you are not able to bring the starting codon, the charged tRNA with the initiating methionine.2525

                      You cannot bring that to the ribosome because this EIF2 GTP complex is required to bring that to the 40S ribosome.2537

                      If you phosphorylate EIF2, you cannot translate anything.2547

                      Another way of global regulation, we have phosphorylate of EIF2,2554

                      another form of global regulation is competitive binding of the 5 prime cap of mRNA.2559

                      Normally, that mRNA, remember, we have a cap.2566

                      That cap is normally bound by EIF4-G.2572

                      Phosphorylated regulatory proteins called 4EBP, binding protein, the cap is normally bound by EIF4-E which is then bound by EIF4-G.2583

                      If these EIF4-E is bound by these 4E binding proteins right, then the EIF4-E will not allow,2614

                      the binding of 4EBP will not allow EIF4-G to bind.2669

                      Therefore, you will not allow translation to occur.2673

                      This can also be used to regulate specific mRNAs but in general, if you affect the binding of initiation factors for translation,2679

                      binding the actual mRNA you can affect all mRNAs that are undergoing translation.2690

                      We can talk about a couple different ways of translational regulation.2698

                      This is what is called translation dependent regulation.2703

                      This is when translation is already started and it is in the elongation phase.2706

                      We have two different types, we have nonsense mediated mRNA decay and we have nonstop mediated mRNA decay.2712

                      Nonsense mediated mRNA decay is when you come across an mRNA that has a premature stop codon.2723

                      What you want to do is degrade those mRNAs so that you do not even have to utilize the energy and2734

                      the amino acids of the whole process to make this short protein which will end up being degraded by the prodiuzome anyway.2741

                      You can degrade mRNAs with the premature stop codon, by removing either the 5 prime cap2751

                      or the 3 prime poly-A tail of the mRNA.2760

                      In which case, you have now freed up either one of those ends to nucleolytic digestion.2764

                      Exonucleases can now come in and just chew it up and degrade it, the mRNA.2771

                      We have another thing called nonstop mediated mRNA decay.2777

                      This occurs when a ribosome is working on an mRNA that wacks a stop codon.2781

                      This can be pretty detrimental to the cell because if there is no stop codon,2789

                      basically the ribosome is stuck with that mRNA attached to it, as well as the protein coming out.2794

                      It just stalled, nothing can happen, you cannot release the polypeptide.2812

                      You cannot separate the ribosome, which means you cannot then translate a new mRNA.2817

                      Without that stop codon, the ribosome is stalled, it cannot do anything.2825

                      We want to be able to fix that.2833

                      This is rescuing, ribosome is translating mRNAs without a stop.2836

                      If you do not have a stop codon, that poly-A sequence in the tail gets translated,2842

                      and that Poly-A, the AAA codon gives you lysine.2849

                      What this does, that causes the ribosome to stall.2854

                      It acts as a signal that there is something wrong.2858

                      What happens is we have eukaryotic releasing factors 1 and 3,2862

                      binding to the ribosome and associating the ribosome from the mRNA.2869

                      At that point, the endonuclease which actually is an unknown endonuclease at this point,2892

                      researchers are still dwelling into this information.2899

                      This endonuclease will degrade the mRNA from 3 prime to 5 prime.2902

                      The protein, if it is enable to fold properly which is very likely due to all of the extra lysines,2908

                      will likely just be signaled to be degraded and be sent to the prodiuzome.2917

                      These are two different ways that you can have translational regulation, actually once translation has occurred.2922

                      Transcriptional regulation and translational regulation, and now I’m on to epigenetics.2936

                      We touched over this a little bit, when we talked about chromatin organization.2942

                      I think it was unit 5 but we are going to talk a little bit about this again, give you a little more detail.2946

                      Epigenetics, what is it?2956

                      That is inherited patterns of gene expression resulting from chromatin alteration.2958

                      It is not in alteration of the DNA sequence that is heritable.2964

                      It is not a change in the base sequence, A, C, T, G.2969

                      But it is a heritable, passing from generation to generation,2974

                      it is a heritable trait of extra non DNA related changes, in terms of the basic cell.2980

                      But it could be due to just small changes, such as a methylation, such as acetylation.2996

                      It can be on the DNA sequence, as well as on the nucleusome itself.3004

                      This can happen, we will talk about three different ways, DNA methylation,3017

                      the nucleosomes or the histone proteins, as well as something called polycomb repressors.3023

                      A very important point of epigenetics is that DNA sequence does not act alone in passing genetic information to future generations.3028

                      Meaning, the methylation state, let us say of DNA or of histone proteins.3039

                      The methylation state usually has to do with what genes are silenced or active, that is able to be sent from generation to generation.3046

                      DNA methylation usually occurs at CPG sites or CG sites.3059

                      The p that just says it is a C right next to a G, only separated by a phosphate which is what is in the backbone of DNA.3064

                      DNA methylation occurs in our CPG sites via the DNA methyltransferase enzyme.3073

                      What you do is you methylate cytosine to get 5-methylcytosine.3081

                      That can keep that methylation even through the next generation.3086

                      However, one drawback to this is that actually spontaneous deamination can turn this 5-methylcytosine into thymine overtime.3092

                      Remember, this cytosine, even as a 5-methylcytosine, will base pair with a guanine.3103

                      If it gets deaminated, it now becomes a thymine.3113

                      On the other strand that was not touched, it is a guanine.3120

                      What you need to do now is, we see that this is in a proper DNA pair, there is a DNA repair that needs to come on.3124

                      If this T is repaired back to a C, no harm no foul.3133

                      If this T is not repaired but the G is repaired to an A, now we have a difference in the original base sequence.3145

                      An epigenetic change has actually turned into a genetic change that gets passed on to next generation.3157

                      This can cause DNA mutations, if not properly repaired.3167

                      CPG islands, I just want to talk about because you will hear that a lot, probably,3173

                      if you read some research papers or if your professors are talking about genetics and especially DNA methylation.3177

                      CPG islands are regions of DNA with the high frequency of CG sites.3184

                      There are usually about 200 base pairs in length and they are associated with promoter regions of a lot of our mammalian genes.3191

                      Usually, when you see CPG island, that is something that should bring to your mind, we are in the promoter of a gene.3199

                      Not all the time, but very frequently.3209

                      Methylation of many of these CPG sites will silence gene transcription,3213

                      leading to heterochromatinization, the formation of heterochromatin.3219

                      The methylation pattern, as I talked about before, it can be passed on to daughter cells.3228

                      This methylation will prevent the binding of a transcription factor called SP1 to the CPG islands.3233

                      The binding of SP1 is part of transcriptional enhancement.3242

                      We also have this other protein involved with DNA methylation called MECP2.3250

                      This is a binding protein of those methylated CPG islands.3257

                      It is a repressor protein that binds to those islands in DNA.3263

                      It will act as a transcriptional repressor.3268

                      It decreases transcription by recruiting histone deacetylase.3272

                      Remember, if you deacetylate histones that usually compacts our chromatin,3275

                      meaning there is no room for transcription of machinery to get in and go through making your mRNA.3281

                      A phosphorylated MECP2 protein has a decreased infinity for methylated CPG sites, and not only to an increase in transcription.3291

                      MECP2 repressor protein, phosphorylate it, it would not bind, therefore, it leads to transcriptional activation.3309

                      SP1 transcription factor, that is an activator protein.3317

                      Let us look at an example using MECP2.3324

                      We have a protein called BDNF, brain derived neurotrophic factor.3329

                      It is a protein implicated in learning a memory class disease.3335

                      Basically, being able to retain your memories and reorganize them.3338

                      This BDNF is release when our dendrites which is a neuro cell, get depolarized, activated.3344

                      BDNF release can lead to a phosphorylation of in MECP2, meaning, it will now not be able to bind the DNA very well,3351

                      meaning you have an increase in transcription.3362

                      On a related but different specifically from this BDNF, if you have a mutation in the gene for the MECP2 protein3368

                      which is also the MECP2 gene, this will cause a disease called rett syndrome.3378

                      This is a syndrome that has clinical manifestations very similar to autism.3385

                      It will affect the neural function.3390

                      In addition to the autism like symptoms, you have stereotypical hand movements3393

                      like handwringing or repeatedly putting your hands in your mouth.3399

                      It is almost unconscious.3403

                      These are some slides that we have already seen in the chromatin organization lecture.3410

                      But I wanted to just have them again so that we can reference them.3416

                      Remember, we a histone core, we have 8 histone proteins, we have 2 dimers of H2 and H2B,3420

                      one tetramer of H3 and H4.3429

                      147 base pairs of DNA wound around.3433

                      It is about 1.65 times around the core.3437

                      We also have linker DNA, 20 to 60 base pairs.3440

                      Just that nucleosome will compact DNA about 6 fold.3446

                      When you add in the linker histone H1, you can compact even more into your 30 nm fiber from your 10nm,3452

                      that will compact DNA about 40 fold.3462

                      You can even compact even more than that, utilizing scaffolding so that you can make a bunch of loops.3466

                      We have talked about the histone code before, there are post translational modifications of our N-terminal histone tails.3477

                      That is part of the epigenetic code.3485

                      If we remember, phosphorylation usually adding a negative charge, acetylation positive charge.3487

                      If you are adding a negative charge that is going to decrease its interaction with DNA.3494

                      It will loosen it, making it more transcriptionally active.3502

                      Acetylation adding positive charge.3506

                      That is neutralizing the negatively charged lysine which is actually going to open you up.3513

                      Methylation will increase the interaction of DNA meaning transcriptional repression.3519

                      You also have ubiquitination which can be binding site for either transciptional activators or repressors.3524

                      We have talked about the histone code being very complicated.3534

                      You can silence, you can have gene expression, gene silencing.3537

                      You can have chromosome condensation, you can have DNA damage repair,3541

                      based on several slight changes in the epigenetic code of the histone.3547

                      We talked about nucleusome assembly.3556

                      When you replicate your DNA, you have to replicate your nucleosomes.3558

                      Some of those nucleosomes get reused in the next generation.3563

                      What those nucleosomes, whatever they had for epigenetic changes, methylations, ubiquitinations, phosphorylations,3569

                      acetylation, whatever, that will be transported with the nucleosome to the daughter cell.3578

                      That can propagate the epigenetic status, from one generation to the next.3587

                      What is very important are those parental H3 H4 tetromers.3600

                      We have talked about the inheritance of the chromatin state.3606

                      Histone acetlytransferases combined acetylated tails using its bromo domain.3611

                      Therefore, spread the acetylation which would decrease the condensation of the heterochromatin,3617

                      to make it more euchromatic, therefore, transcriptionally active.3631

                      Or you can have the same thing happening with histone methyltransferases3636

                      binding methylated regions via its chromo domain, causing a condensation.3641

                      Therefore, more heterochromatization IE transcriptional repression.3649

                      We will just write this real quick.3657

                      Histone acetyltransferase binds acetyl groups via its bromo domain leading to a looser state3660

                      which is more likely going to be a transcriptional activation.3679

                      We have histone methyltransferase via its chromo domain, binding methylated histone tails3685

                      which will tighten, condense, leading to a more likely repressed state.3698

                      Chromatin remodeling, it is all part of epigenetics.3711

                      As I said on the previous slide, when we have histone acetyltransferase,3715

                      as well as chromatin remodelers like the swi/snf complex,3718

                      that allows you to loosen up the interaction of DNA with the histones and allow your transcription of machinery to come in.3722

                      Therefore, being in an activated state.3732

                      If you have your histone deacetylases and your histone methyltransferases, that is going to cause a tighter association with DNA.3735

                      Therefore, less room for your big RNA polymerase and all your initiation factors to come in and allow transcription.3743

                      This is a transcriptionally repressed state.3751

                      Our last example, I believe, we have methylation of DNA and histones seeming to be correlated.3757

                      Usually, when both DNA and histone tails are methylated, this will be our transcriptionally repressed state.3765

                      Acetylation of histones leads to loosening of DNA around the histone, offering a high likelihood of replication or transcription.3773

                      Acetylation, high likelihood of replication and transcription.3784

                      Methylation, transcriptionally repressed state.3792

                      Finally, just as a mentioned, we have our polycomb repressors.3801

                      These polychrome repressors just are a family of proteins that can remodel chromatin,3806

                      such that epigenetic silencing will take place.3813

                      PRC, that is just polycomb repressor complex or repressive complex.3818

                      PRC2 protein complex will bind DNA and try methylate your histone 3 at lysine 27.3825

                      This will cause a repression.3834

                      The PRC1 protein complex will also repress transcription.3841

                      We have another protein complex called the MLL protein complex which will reverse this repression by demethylating H3K27.3846

                      A couple things I want to point out is that, the PRC1 protein complex, a mutation in this,3859

                      if you are a mutant, like they can make in labs, mutants die perinatally.3870

                      Meaning, either at the time of birth or shortly after no more than two weeks, in under a couple of weeks.3879

                      The PRC2 mutants are what are called embryonic lethal, meaning they never get born in the first place.3896

                      These are really important complexes.3913

                      One more thing to show you how important they are is that, over expression, if PRC1 and PRC2 are too active,3915

                      either too many around, they are repressing too much.3925

                      If they are over expressed that is actually correlated with a more severe and more invasive types of cancer.3927

                      Your cancer is likely to be, you are worse off if you have over expression of your polycomb repressor complexes.3942

                      That is the end of the lesson today, I hope you enjoyed our lesson.3952

                      I hope I see you back for our next one.3957

                      Thank you for joining us at, I hope to see you again.3963