Lecture 12 Tr Reg Gene Exp-II PDF

BIOL 366 Lectures 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 le...

BIOL 366 Lectures 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 homeodomain 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. FIGURE 19-17 The DNA-binding domain of the bacterial Lac repressor (as a ribbon structure) interacting with the major groove of DNA. • The h-t-h motif is generally part of a somewhat larger DNA-binding domain. 7 The homeodomain DNA-binding motif • Was first identified in fruit fly • Is made up of 60 aa sequence • Is found in TF that regulate body pattern development. • Is found in proteins from a wide variety of multicellular organisms, including humans. FIGURE 19-18. Paired , a Drosophila TF, uses homeodomain DNA-binding motif. 8 The homeodomain DNA-binding motif. • Has a helix-turn-helix motif that is unique - It is composed of three α-helices - Only 2 of these helices (2 & 3) correspond to the well known “helixturn-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. 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 (conserved region) ➢ 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 FIGURE 19-20: The b-hlh motif of the human Max TF bound to its DNA target 12 • 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 human Max TF bound to its DNA target 13 The zinc finger motif Proteins with this domain have diverse functions, including roles in: • DNA recognition • RNA packaging • transcriptional activation • protein folding • lipid binding 14 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 15 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. 16 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 FIGURE 19-21 17 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). 18 Utility of DNA binding proteins in designing nuclease for gene editing 19 ZFNs (zinc-finger nuclease ) Modified from Fig 2 in Carol, 2012 • In these “engineered” nucleases, zinc fingers are joined to a FokIderived DNA cleavage domain (large oval) by a short linker o FokI is a type II restriction endonuclease found in bacterium Flavobacterium okeanokoites FokI cleavage site o FokI contains an N-terminal DNA-binding domain and a C-terminal DNA cleavage domain. 20 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 - Zinc fingers direct the complex to a specific site - FokI cleaves the target DNA 21 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 Modified from Fig 2 in Carol, 2012 22 TALENs (transcription activator–like effector nucleases) • 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 Modified from Fig 2 in Carol, 2012 23