Lecture 12 Tr Reg Gene Exp-II PDF

Summary

This document provides a concise description of transcriptional regulation of gene expression. It details different DNA-binding motifs including helix-turn-helix, homeodomain, leucine zipper, basic helix-loop-helix, and zinc finger motifs. The document also gives examples of proteins using these motifs, and their roles in gene expression.

Full Transcript

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

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