RR5-Proteins that regulate transcription-2023.pdf

McGill BIOL200 - Fall 2023 Proteins that regulate transcription-Activators © R. Roy, 2023 McGill BIOL200 - Fall 2023 Most eukaryotic genes are regulated by multiple transcriptional control elements © R. Roy, 2023 McGill BIOL200 - Fall 2023 Cis-acting regulatory sites can be found through...

McGill BIOL200 - Fall 2023 Proteins that regulate transcription-Activators © R. Roy, 2023 McGill BIOL200 - Fall 2023 Most eukaryotic genes are regulated by multiple transcriptional control elements © R. Roy, 2023 McGill BIOL200 - Fall 2023 Cis-acting regulatory sites can be found through linker scanning mutations Reporter genes-relative quantification of transcriptional efficacy Some common “Reporters” -GFP -β-galactosidase (lacZ) -thymidine kinase (tk) -luciferase (luc) -chloramphenicol acetyltransferase (CAT) subregions of the promoter required for activation of transcription © R. Roy, 2023 McGill BIOL200 - Fall 2023 Electrophoretic mobility shift assays (EMSA)- DNA binding activity • EMSA or gel/band/mobility shift assays to assay for DNA binding activity • A radiolabelled dsDNA segment is used as a probe • Protein:DNA mobility is altered in the non-denaturing polyacrylamide gel • EMSA/Gel Shifts cannot reveal the precise sequence that is bound by the protein!!! © R. Roy, 2023 McGill BIOL200 - Fall 2023 An assay to test DNA binding transcription factors CANDIDATE PROTEIN cDNA LacZ reporter Identified Control Element Co-transfection of Plasmid 1 and 2 CANDIDATE PROTEIN © R. Roy, 2023 McGill BIOL200 - Fall 2023 Transcription factors recognize specific DNA sequence motifs alpha-helical domain, the so-called RECOGNITION HELIX. recognition helices bacteriophage repressor 434 homodimer Binding occurs through noncovalent interactions with atoms in the bases. interaction with the major groove of DNA. © R. Roy, 2023 McGill Transcription factors are modular most transcription factors have multiple domains that each perform distinct functions BIOL200 - Fall 2023 GAL4 an example: the GAL4 transcription factor from yeast -> contains a DNA binding domain to bind UASGAL -> contains an activation domain to stimulate transcription © R. Roy, 2023 McGill BIOL200 - Fall 2023 Modular structure is very common in eukaryotic transcription factors transcription factors can possess domains for: DNA binding transcription activation transcription repression chromatin remodelling nuclear import protein interaction © R. Roy, 2023 McGill BIOL200 - Fall 2023 Homeodomain proteins ➲ The homeodomain was named due to its presence in several transcription factors that give rise to homeotic transformations when mutated at particular residues © R. Roy, 2023 McGill BIOL200 - Fall 2023 Homeodomain proteins ➲ The homeodomain was named due to its presence in several transcription factors that give rise to homeotic transformations when mutated at particular residues homeodomain binding sites © R. Roy, 2023 McGill BIOL200 - Fall 2023 Different types of zinc finger DNA binding domains exist C2H2 types usually contain three or more finger units and bind to DNA as monomers C4 types usually contain only two finger units and bind to DNA as homo- or heterodimers ie…steroid hormone receptors the C6 Zinc Finger transcription factor is a variation wherein six cysteine metal ligands coordinately bind two Zn2+ ions © R. Roy, 2023 McGill BIOL200 - Fall 2023 Leucine zipper proteins -Leucine zipper proteins bind DNA exclusively as homo- or heterodimers with their extended alpha-helices, which bind the major groove of the DNA -they contain a leucine or a different hydrophobic amino acid in every seventh position in the C-terminal region of the DNA binding domain… bZIP proteins -these hydrophobic residues form a coiled coil domain, which is required for dimerization. cmgm,stanford.edu © R. Roy, 2023 McGill BIOL200 - Fall 2023 Helix-Loop-Helix proteins -helix-loop-helix (HLH) proteins are very similar to leucine zipper proteins, however instead of an extended alpha-helix they are characterized by two alpha-helices, which are connected by a short loop -HLH proteins contain hydrophobic amino acids spaced at intervals characteristic of an amphipathic alpha-helix in the C-terminal region of the DNA binding domain © R. Roy, 2023 McGill BIOL200 - Fall 2023 Transcription factors of unrelated classes can also bind COOPERATIVELY protein-protein interaction favours formation and stability of the ternary complex (Cooperative DNA Binding) © R. Roy, 2023 McGill BIOL200 - Fall 2023 Combinatorial possibilities greatly extend the potential for diversified gene regulation The combination of transcription factor binding sites in promoters leads to a diversity of transcriptional responses Homo- and heterodimer formation is common among transcription factors ie…three transcription factors that can homodimerize or heterodimerize => 6 different possible combinations © R. Roy, 2023 McGill BIOL200 - Fall 2023 To what DNA sequences are transcription factors bound? ChIP-Chromatin Immunoprecipitation (ChIP-seq) -crosslink macromolecules with formaldehyde -shear the DNA into small fragments -immunoprecipitate with an antibody -NG sequencing of bound DNA -DNA will correspond to genes that are bound by transcription factor © R. Roy, 2023 McGill BIOL200 - Fall 2023 DNA binding elements can be determined © R. Roy, 2023 McGill BIOL200 - Fall 2023 The Mediator is yet another BIG multisubunit protein complex © R. Roy, 2023 McGill BIOL200 - Fall 2023 DNA-bound activators interact with a single Mediator complex The multisubunit Mediator complex can bridge vast sections of chromatin to enhance transcriptional initiation. Head Middle This is mediated through associations between various transcription factor activation domains and specific Mediator subunits. Tail © R. Roy, 2023 McGill BIOL200 - Fall 2023 Transcription does not occur in a linear manner…. The role of the Mediator is consistent with the topological hierarchy observed in transcriptionally active looped-out chromatin. It likely “mediates” the effects of enhancer elements and their binding proteins on the basal/general transcription machinery (RNA Pol II). © R. Roy, 2023