BIOL 366 Lecture 12: Transcriptional Regulation of Gene Expression PDF

BIOL 366 Lecture 12 Transcriptional regulation of gene expression: 1) Text Section: 19.1 – 19.2 2) Article A CRISPR Approach to Gene Targeting by Dana Carroll (doi:10.1038/mt.2012.171) Key Terms: transcription factor, activator, recognition helix, helix-turn-helix mo...

BIOL 366 Lecture 12 Transcriptional regulation of gene expression: 1) Text Section: 19.1 – 19.2 2) Article A CRISPR Approach to Gene Targeting by Dana Carroll (doi:10.1038/mt.2012.171) Key Terms: transcription factor, activator, recognition helix, helix-turn-helix motif, homeodomain motif basic leucine zipper motif, basic helix-loop-helix motif, zinc finger motif, TALE, TALEN, 1 Eukaryotic promoters use more regulators than bacterial ones Bacterial promoters: - are typically near, or overlap, the coding region - are usually regulated by only one or two regulatory proteins Fig 19-13 (a) 2 Eukaryotic genes, especially those of multicellular organisms: - usually have numerous regulator-binding sites, which - can span a large region (more than 50 kb) - can be upstream and downstream from the promoter - can be within the coding sequence of the gene itself Fig 19-13 (b) 3 Transcription Factors (TF): Gene activation and repression in both bacteria and eukaryotes require transcription factors (also called transcription regulators). TFs: Proteins that affect the regulation and transcription initiation of a gene by binding to a regulatory sequence near or within the gene and interacting with RNA polymerase and/or other transcription factors. TF binding sites usually contain inverted repeats (a nucleotide sequence, followed by the reverse, complementary sequence) FIGURE 19-16 4 Notes on Transcription Factors (TF): The recognition helix: The recognition of DNA by a TF is typically through certain amino acid side chains of an α helix referred to as the recognition helix. FIGURE 19-17 The DNA-binding domain of the bacterial Lac repressor (as a ribbon structure) interacting with the major groove of DNA. 5 TFs are classified based on the presence of specific conserved motifs: - The helix-turn-helix DNA-binding motif - The leucine zipper motif - The basic helix-loop-helix motif - The zinc finger motif 6 The helix-turn-helix (h-t-h) DNA-binding motif. Many bacterial and eukaryotic regulatory proteins use the h-t-h motif for DNA binding The h-t-h motif consists of about 20 amino acids (aa) The 20 aa form two short α helices connected by a β turn. The h-t-h motif is generally part of FIGURE 19-17 The DNA-binding a somewhat larger DNA-binding domain of the bacterial Lac repressor domain. (as a ribbon structure) interacting with the major groove of DNA. 7 The homeodomain; A unique h-t-h type motif - Is composed of three α-helices - Only 2 of these helices (2 & 3) correspond to the well known “helix-turn-helix” motif - The N-terminal residues of the homeodomain reach around the DNA duplex and interact with the minor groove of DNA (chromosome) FIGURE 19-18. Paired , a Drosophila TF, uses homeodomain DNA-binding motif. 8  The homeodomain DNA-binding motif Is found in TFs that regulate body pattern development Was first identified in fruit fly Also found in proteins from a wide variety of multicellular organisms, including humans In plants, they contribute to floral organ (petal, sepal, anther, etc.) development FIGURE 19-18. Paired , a Drosophila TF, uses homeodomain DNA-binding motif. 9 The basic leucine zipper motif: Is made up f 60 – 80 amino acids, depending on species Is an amphipathic α helix, with a series of hydrophobic aa residues concentrated on one side of the helix. Is found in many eukaryotic and a few bacterial regulatory proteins 10 The basic leucine zipper motif, continued. A striking feature of this α helix is the occurrence of Leu residues at every seventh position, forming a hydrophobic surface along one side of the helix, where two identical subunits dimerize. There are basic residues (Lys (K) and Arg (R)) in the DNA-binding region. Leucine zipper helices are primarily used only for dimerization; separate motifs are used for DNA binding. Fig 19-19 11 The basic helix-loop-helix (b-hlh) TFs motif Is made up of ~ 50 amino acids Contains two amphipathic α helices, joined by a loop of variable length One helix contains basic residues and mediates DNA binding One helix does NOT contain basic residues and mediates dimer formation TF with b-hlh motif are often involved in development or cell cycle activity. Examples c-Myc and HIF-1, which have been linked to cancer FIGURE 19-20: The b-hlh motif of the 12 human Max TF bound to its DNA target The zinc finger motif The zinc finger domain consists of ~30 amino acids The domain forms an elongated loop stabilized by Zn2+ ions – Zn2+ ions do not directly interact with the DNA They are found in eukaryotic and bacterial proteins (few) There are many types; Type I and II are most common 13 The zinc finger motif Single Zinc figure interactions are weak Many DNA-binding proteins have multiple zinc fingers - 37 in one Xenopus DNA-binding protein Multiple zinc fingers strengthen binding to DNA Proteins with multiple zinc fingers have been designed for gene editing. 14 Type I zinc finger motif - Are among few regulatory proteins that function as monomers - DNA binding site is long and has no internal inverted repeats - Uses ONE Zn2+ ion to stabilize the DNA binding domain Example: Mouse Zif268 protein 15 FIGURE 19-21  Type II zinc finger motifs - Combine “Zn2+-binding” motif with the “helix-turn-helix” motif - Uses two Zn2+ ions to stabilize the DNA binding domain - The DNA binding domain has a h-t-h motif - Bind DNA as dimers, using a leucine zipper to mediate dimer contacts Example: The yeast Gal4 protein (Figure 19-22). 16 The zinc finger motif Proteins with this domain have diverse roles in: DNA recognition RNA packaging transcriptional activation protein folding lipid binding 17  Utility of DNA binding proteins in designing nuclease for gene editing 18  ZFNs (zinc-finger nuclease ) Modified from Fig 2 in Carol, 2012 In these “engineered” nucleases, zinc fingers are joined to a FokI- derived DNA cleavage domain (large oval) by a short linker o FokI is a type II restriction endonuclease found in the bacterium Flavobacterium okeanokoites FokI cleavage site 19  ZFNs (zinc-finger nuclease ) Modified from Fig 2 in Carol, 2012 Each zinc finger (small ovals) in a zinc-finger nuclease (ZFN) binds primarily to three consecutive base pairs A minimum of three fingers are required to provide sufficient affinity for DNA - Zinc fingers direct the complex to a specific site - FokI cleaves the target DNA 20 TALENs (Transcription Activator–Like Effector Nucleases) TALENs are TALEs (transcription activator-like effectors) fused to FokI nucleases TALEs are secreted by Xanthomonas bacteria when they infect plants. They bind plant DNA sequences through a ~34 aa central repeat domain TALEs target the nuclease complex to specific DNA site FokI cleaves DNA 21 Modified from Fig 2 in Carol, 2012 TALENs (continued) Each TALE module (small ovals) binds a single base pair The minimum effective number of modules is 10–12, but more are typically used. The linker to the FokI domain (large ovals) is longer than for ZFNs and contains additional TALE-derived sequences It is much easier to design specific TALENs than ZFNs 22 Modified from Fig 2 in Carol, 2012