Test 4 Review

Transcriptional Regulation Prokaryotes Conclusion from Movie In the absence of regulatory proteins, RNA is often expressed at a low, Basal level An activator increases the lever of transcription Activation can occur by recruitment or by allostery A repressor decreases the level of transcription The sites on DNA where a repressor binds is called an operator. Activation Recruitment CAP (Lac Operon) Allosteric Allolactose binds the lac repressor and changes the shape and releases the lac repressor cAMP brings to CAP to induce a change in shape of CAP to allow cap to bind to DNA NtrC activate transcription of glnA (Nitrogen metabolism) MerR activate transcription merT (Twists DNA) Repression Why can a wild-type ? phage not grow on a ? lysogen? In order to maintain the lysogenic state, a lysogen constantly produces repressor protein. This repressor protein is then available to immediately bind OL and OR of any ? that enters the cell, thereby blocking the lytic pathway. Will a cI– mutant (that is, a ? repressor mutant) of ? grow in a ? lysogen? Why or why not A cI mutant will not grow on a ? lysogen, because the repressor protein that is already produced by the lysogen will be sufficient to bind OL and OR sites in the mutant and thereby prevent the mutant phage from pursuing the lytic pathway. Thus, even though the cI mutant cannot form a stable lysogen, it cannot grow lytically on a lysogen either. Mutations that allow ? to grow in a ? lysogen are called virulent (vir) mutations. To which type of lac operon mutation are virulent mutations similar? Virulent mutations are analogous to the constitutive, cis-acting lac mutations—that is, the mutations within the operator that prevent Lac repressor from binding. It turns out that virulent mutations of ? are very hard to isolate because they are extremely rare, occurring in about one of every 1015 phages. Can you think of a reason why such mutants are so rare? Virulent mutations are rare in ? because they must eliminate repressor binding at both OL & OR Transcriptional Regulation Eukaryotes Eukaryotic forms of regulation Splicing Transcript stability RNA transport Initiation of transcription Protein stability Post translational modifications of proteins Protein transport Compare and contrast the DNA sequence elements involved in regulating transcription in bacterial cells and eukaryotic cells. Bacterial cells and eukaryotic cells include promoter sequences within the DNA upstream of the coding sequence of a gene. Bacterial and eukaryotic cells also include DNA-binding sites for regulatory proteins like repressors or activators. One activator and/or one repressor protein typically control bacterial genes, whereas the regulatory elements of eukaryotic genes can be more elaborate. In eukaryotic cells, more regulatory elements may be present, the regulatory elements may be upstream or downstream of the promoter, and the regulatory elements may include binding sites for multiple activators and/or repressors. Multiple regulatory elements are grouped as enhancers in multiple organisms and insulator or boundary elements may be present. The regulatory elements of eukaryotic genes may also be located at much greater distances form the gene they regulate than is the case in bacteria. Which of the following protein domains would be most likely to recognize and bind to acetylated lysine residues on nucleosomes? Acelylation creates specific binding sites for bromodomain-containing proteins. A component of TFIID contains a bromodomain that allows it to bind with more efficiency to acetylated nucleosomes than to unacetylated nucleosomes. Thus, a gene bearing acetylated nucleosomes at its promoter will be more likely to recruit transcriptional machinery than one with unacetylated nucleosomes. Which one of the following gene families is not under the control of a group of regulatory elements called a locus control region (LCR) or a global control region (GCR)? A) human globin genes B) mouse globin genes C) mouse HoxD gene cluster D) S. cerevisiae mating-type genes The mating-type genes in yeast are under combinatorial control and do not utilize an LCR or a GCR. The three cells types of yeast (haploid a, haploid , and diploid a/) are determined by gene expression within the mating-type locus. Various combinations of different regulatory proteins (a1, 1, & 2, and Mcm1) work together to regulate the cell type. These regulatory proteins function as activators or repressors, depending on the various combinations bound at the MAT locus. You isolate a mutant strain of mice that grow at an unusually fast rate, perform a blood test on the mice, and find that they have elevated levels of insulin-like growth factor. The phenotype is the result of a mutation in a region of the genome containing a gene encoding a DNA methylase. What kind of mutation is causing the rapid growth of these mice? The mice are probably growing abnormally fast—and producing higher levels of insulin-like growth factor—because they express both copies of the Igf2 gene. Normally, the maternal copy of the gene (that is, the copy present on the chromosome inherited from the mother) is shut off, because an insulator element located near the gene prevents a nearby enhancer from activating transcription. The paternal gene, in contrast, is normally expressed, because the insulator element on the paternal chromosome is methylated and thereby prevented from interfering with the action of the enhancer. The mutation, then, could be a mutation in a methylase enzyme that causes the enzyme to methylate the insulator element on both the maternal chromosome and the paternal chromosome. Accordingly, both the maternal and the paternal Igf2 genes would be expressed, leading to an abnormally high amount of growth factor production in cells and producing faster-than-normal growth. Alternatively, the mutation could also cause upregulation of one allele, either the maternal or paternal allele, for Igf2. Describe 5 ways activators can activate a gene Recruitment Activators can activate transcription by recruiting other parts of the transcriptional machinery to the promoter, either the polymerase in prokaryotes, or other factors that interact with the polymerase in eukaryotes (no direct recruitment of polymerase in eukaryotes). Examples CAP w/pol in lac, NtrC AraC cI in prokaryotes GAL 4 in yeast HSP70 in Drosophila in eukaryotes. Note that TFIID is NOT an activator-it is the thing recruited by the activator. Conformational Change in the DNA An activator can induce a conformational change in the DNA that will allow other activator binding sequences to come near the promoter and induce transcription. This mechanism occurs in both prokaryotes & eukaryotes. Examples Mer in prokaryotes Interferon regulation in eukaryotes Conformational Change in the Pol/Transcription Machinery Activators can induce gene expression by causing a conformational change in the polymerase/transcription machinery (from open to closed) and this occurs in both pro and eukaryotes. Example NtrC’s ATPase activity that changes the confirmation of the polymerase Recruit Nucleosome modifiers Activators can also activate gene expression by recruiting nucleosome modifiers to alter the chromatin structure. A change in structure can uncover DNA binding sites for access by the transcriptional machinery. Example SBF and SWI5 with the HO gene this is specific to eukaryotes. Histone Tails to be Acetylated Activators can allow the histone tails to be acetylated thereby opening up the DNA, or create sites for bromodomain proteins. This occurs only in eukaryotes as prokaryotes do not have histones. Example MIG1 and SAGA in the GAL system in yeast is an example. List four mechanisms for repression in eukaryotes Competition The repressor binds to a site that partially overlaps with the site the activator binds to, so that the two molecules compete for the same site. Example Inhibition Although they bind to different sites, the repressor prevents the activator from working by physical contact of the repressor with the activator. Example Direct repression The repressor binds to a component of the transcription complex, usually the mediator, to prevent the polymerase from leaving the promoter Example Indirect repression Repression by recruiting histone modifiers that alter the nucleosomes in ways to prevent transcription (deacetylation, methylation). Example Mig1 repressing GAL1 in yeast Activation of HO Gene The HO Gene Codes of Ho endonuclease Is only expressed in mother cells Is only expressed at a certain point in the cell cycle SWI5 Is only activated in the mother cell Binds multiple sites >1kb from the HO promoter Recruits nucleosome modifiers which reveal SBF binding sites SBF IS only active at the correct stage of the cell cycle Recruits the Mediator, Activating HO expression Regulatory RNA’s What are three ways prokaryotes can regulate at the level of translation? Blocking the ribosome binding site with a protein; hiding the ribosome binding site via 2ndary structure of the message; allosteric change in sigma factor (sigma 54 in optional readings) Why won’t these work in eukaryotes? How do eukaryotes regulate translation? Eukaryotic ribosomes don’t have a binding site-they find the 5’ cap and scan. General mechanisms for regulation include a 5’cap binding protein that prevents eIF4G from working; and phosphorylation of eIF2 which keeps GTP exchange from happening (in the initiator tRNA binding to the 40S subunit) Compare how prokaryotes & eukaryotes deal with premature stops or lack of stops and “bad messages” Because the ribosome would be stuck at the end of a prokaryotic message that had no stop codon, prokaryotes use a special molecule SsrA, a “transfer-like” RNA that adds an alanine to the peptide, then provides 10 aa worth of code & a stop to remove the ribosome. This code tags the protein for degradation. Premature stops are not controlled. In contrast, eukaryotes identify messages with premature stops by the presence of SR, EXE, and other proteins that should be removed in translation; if translation through the end of the message does not occur, it triggers Upf proteins and deadenylating enzymes which destroy the message. If there is no stop codon, when the ribosome hits the poly a sequence, it will add a string of lysines, marking the protein for destruction. Which of the following statements about riboswitches is true? Riboswitches control gene expression through changes in RNA secondary structure. Knowing how the CRISPR/Cas system works in E. coli, how could you engineer it for knocking out genes? Since the CRISPR/Cas system uses a homologous piece of RNA to target foreign DNA for cleavage, creating a synthetic CRISPR/Cas system with the sequence of the gene one wishes to mutate and expressing it in a eukaryotic cell causes a break in that gene, repair of which sometimes creates a targeted gene mutation In which of the following pre-mRNA sequence elements can pre-miRNAs be coded? exons, introns, and noncoding regions Which of the following proteins combine to form the Microprocessor complex used to produce active miRNA? Pasha (or DGCR8) and Drosha What is the function of the PAZ domains in Dicer and Argonaute proteins? In both proteins, the PAZ domains recognize and bind the 3 end of a double-stranded RNA molecule. Which of the following is a shared characteristic of miRNAs and siRNAs? miRNAs and siRNAs are both processed in the cytoplasm by the protein Dicer. Which of the following statements about the function of regulatory RNAs in X-chromosome inactivation is NOT true? Xist RNAs produced by one X-chromosome can initiate inactivation of genes located on the other X-chromosome (they are trans-acting regulators). Gene Regulation in Development and Evolution What is a morphogen? Name at least three molecules described in this chapter that could be considered morphogens. What kind of experiments or observations could be carried out to determine if a given molecule acts as a morphogen? A morphogen is a secreted signaling molecule produced by cells in one region of a tissue or organism. As the morphogen diffuses away from the morphogen-producing cells, it conveys positional information on cells. Morphogens are diffusable elements that generate a concentration gradient, and cells adopt particular fates depending on the concentration of morphogen they are exposed to. Examples of morphogens discussed in the chapter include Sonic hedgehog, Dorsal, Bicoid, and Hunchback. Because morphogens work by causing cells to adopt different fates depending on how much of the morphogen they are exposed to, a morphogen must be present as a gradient in order to work (although, in some cases, the gradient can be something other than overall concentration, such as the gradient of nuclear localization seen with Dorsal). Therefore, in order to determine if a particular molecule is a morphogen, one important thing to do would be to visualize its distribution and confirm that it indeed forms a gradient. In addition, you could confirm that different concentrations of the molecule cause cells to adopt different fates. For example, if a putative morphogen directs cells to take on three potential fates—with cells closest to the morphogen adopting cell fate A, cells at a medium distance adopting cell fate B, and with the most distant cells developing into fate C— then you would expect that a uniform concentration of the morphogen throughout the tissue might cause all of the cells to adopt cell fate A. Similarly, mutations that eliminate morphogen expression altogether might make all of the cells adopt fate C. Finally, you could carry out experiments in which the gradient is altered, for example, by mildly overexpressing the morphogen from its normal source. In this case, you would expect to shift the boundary between the A and the B cells (and between the B and C cells) so that it is now located farther away from the source of the morphogen. Which of the following can regulate gene expression during development, but does NOT represent a pathway of cell-to-cell signal transduction or establishment of an extracellular gradient? mRNA localization A ligand binding to a cell surface receptor triggers phosphorylation of DNA-binding proteins. A ligand binding to a cell surface receptor triggers release of DNA-binding proteins. morphogen secretion by a cluster of cells Three strategies have evolved for instructing cells to express specific genes during development: cell-to-cell contact, signaling through the diffusion of a secreted signaling molecule (morphogen), and localization of maternal mRNAs in an egg (either before or after fertilization). Only the third listed process—mRNA localization—does not represent a pathway of cell-to-cell signal transduction or establishment of an extracellular gradient for regulating gene expression. Localization of mRNAs allows cells to distribute critical regulatory molecules asymmetrically to specific parts of a cell, typically via transport along elements of the cytoskeleton. Drosophila Embryogenesis After fertilization, the nucleus undergoes a series of 10 rapid cleavages Most of the Nuclei migrate to the periphery of the cell After three more cleavages, the nuclei are densely packed around the edge of the cell Cell membranes from, transforming the egg into a cellular blastoderm Localization of each nucleus determines its fate Localized mRNA, Such as the Oskar mRNA, can help determine call fate Concentration gradients, Such as the gradients of Dorsal and Spatzle, can also determine cell fate. Why do many genes involved in patterning of the embryo have especially large regulatory regions? Patterning genes have such large regulatory regions so that they can accommodate different combinations of the large number of DNA-binding proteins. The number of unique possible combinations of binding proteins help produce the precise and complex gene expression patterns that are required for the development of an animal. Examples See next slide for an example-eve is expressed in seven stripes in the embryo, whose positions are determined by particular combinations of various regulatory molecules. Specifically, the eve promoter contains five different enhancers that together produce the seven stripes of eve expression (see figures below). The stripe 2 enhancer, for example, contains binding sites for Bicoid, Hunchback, Giant, and Krüppel. Bicoid and Hunchback activate expression within the stripe, and in fact are present at high enough levels to activate the stripe 2 enhancer throughout the anterior half of the embryo. The domain of expression is restricted to the stripe, however, because Giant and Krüppel act as repressors to define its anterior and posterior boundaries, respectively. Krüppel, which is expressed in a broad stripe in the middle of the embryo, represses eve transcription by binding to the enhancer at sites overlapping Bicoid binding sites, and also by "quenching" the activity of any Bicoid that does bind. Giant, which is present at high levels in the anterior part of the embryo, presumably blocks eve expression to the anterior side of the stripe using a similar mechanism. Spatzle is laid down between the membrane and the shell of the Drosophila egg in a gradient by the mother through the nurse cells. This means the concentration of Spatzle is higher in the ventral side of the egg. Spatzle is cleaved upon fertilization by a protease and the resulting ligand binds to the Toll receptor which is equally distributed throughout the embryo. More Toll binding occurs in the ventral portion because the concentration of Spatzle is higher. Toll then acts on Pelle and Tube (this is a phosphorylation). Pelle and Tube cause Cactus to be degraded, releasing Dorsal from the cytoplasm into the nucleus. So the amount of Dorsal in the nucleus is related to the amount of Spatzle in the extra cellular space of the egg, resulting in a gradient of Dorsal in the nuclei that increases in the ventral nuclei High concentrations of Dorsal activate twist expression in the most ventral portions, because twist has low affinity binding sites for Dorsal. Rhomboid and Sog, which have medium and high affinity sites for Dorsal also have their expression activated in a larger area related to the Dorsal gradient, with sog activated in the outermost cells and rhomboid and sog in cells closer to the ventral end. However, neither rhomboid nor sog is activated in the most ventral region with twist because Twist and Dorsal together activate snail. Snail is a repressor of rhomboid and sog. Thus the end result is a “code” of twist, rhomboid and sog expression telling the cells where they are in relation to the ventral side, all based on the Spatzle deposited when the egg was laid. Which of the following is an accurate description of synteny? A block of genes linked together on the same chromosome that is conserved between distantly related animals Comparative genome analysis has revealed that distantly related animals share conserved blocks of linked genes (show a high degree of synteny). Even organisms that have not had a common ancestor for hundreds of millions of years can exhibit some synteny.