Lecture Molecular biology (Fifth Edition): Chapter 12 - Robert F. Weaver

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The main contents of this chapter include all of the following: Categories of activators, structures of the DNA-Binding motifs of activators, independence of the domains of activators, functions of activators, interaction among activators, regulation of transcription factors.

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Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 12 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 12 Transcription Activators in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transcription Activators of Eukaryotes • The general transcription factors by themselves dictate the starting point and direction of transcription but they are capable of sponsoring only a low level of transcription or basal transcription • Transcription of active genes in cells rises above the basal level • Eukaryotic cells have additional, gene-specific transcription factors called activators that bind to DNA elements called enhancers to provide the extra needed boost to transcription 12-2
12.1 Categories of Activators • Activators can stimulate or inhibit transcription by RNA polymerase II • Structure is composed of at least 2 functional domains – DNA-binding domain – Transcription-activation domain – Many also have a dimerization domain 12-3
DNA-Binding Domains • Protein domain is an independently folded region of a protein • DNA-binding domains have DNA-binding motif – Part of the domain having characteristic shape specialized for specific DNA binding – Most DNA-binding motifs fall into 3 classes; zinc-containing modules, homeodomains and bZIP and bHLH motifs 12-4
Zinc-Containing Modules • There are at least 3 kinds of zinc- containing modules that act as DNA- binding motifs • All use one or more zinc ions to create a shape to fit an -helix of the motif into the DNA major groove – Zinc fingers – Zinc modules – Modules containing 2 zinc and 6 cysteines 12-5
Homeodomains • These domains contain about 60 amino acids • Resemble the helix-turn-helix proteins in structure and function • Found in a variety of activators • Originally identified in homeobox proteins regulating fruit fly development 12-6
bZIP and bHLH Motifs • A number of transcription factors have a highly basic DNA-binding motif linked to protein dimerization motifs – Leucine zippers – Helix-loop-helix • Examples include: – CCAAT/enhancer-binding protein – MyoD protein 12-7
Transcription-Activating Domains • Most activators have one of these domains • Some have more than one – Acidic domains such as yeast GAL4 with 11 acidic amino acids out of 49 amino acids in the domain – Glutamine-rich domains include Sp1 having 2 that are 25% glutamine – Proline-rich domains such as CTF which has a domain of 84 amino acids, 19 proline 12-8
Summary • Eukaryotic activators are composed of at least two domains: a DNA-binding domain and a transcription-activating domain • DNA-binding domains contain motifs such as zinc modules, homeodomains, and bZIP or bHLH motifs • Transcription activating domains can be acidic, glutamine-rich or proline-rich 12-9
12.2 Structures of the DNA-Binding Motifs of Activators • DNA-binding domains have well-defined structures • X-ray crystallographic studies have shown how these structures interact with their DNA targets • Interaction domains forming dimers, or tetramers, have also been described • Most classes of DNA-binding proteins can’t bind DNA in monomer form 12-10
Zinc Fingers • Described by Klug in GTF TFIIIA • Nine repeats of a 30-residue element: – 2 closely spaced cysteines followed 12 amino acids later by 2 closely spaced histidines – Coordination of amino acids to the metal helps form the finger-shaped structure – Rich in zinc, enough for 1 zinc ion per repeat – Specific recognition between the zinc finger and its DNA target occurs in the major groove 12-11
Arrangement of Three Zinc Fingers in a Curved Shape The zinc finger is composed of: – An antiparallel -strand that contains 2 cysteines – 2 histidines in an -helix – Helix and strand are coordinated to a zinc ion 12-12
The GAL4 Protein • The GAL4 protein is a member of the zinc- containing family of DNA-binding proteins • Each GAL4 monomer contains a DNA- binding motif with: – 6 cysteines that coordinate 2 zinc ions in a bimetal thiolate cluster – Short -helix that protrudes into the DNA major groove is the recognition module – Dimerization motif with an -helix that forms a parallel coiled coil as it interacts with the -helix on another GAL4 monomer 12-13
The Nuclear Receptors • A third class of zinc module is the nuclear receptor • This type of protein interacts with a variety of endocrine-signaling molecules • Protein plus endocrine molecule forms a complex that functions as an activator by binding to hormone response elements and stimulating transcription of associated genes 12-14
Type I Nuclear Receptors • These receptors reside in the cytoplasm bound to another protein • When receptors bind to their hormone ligands: – Release their cytoplasmic protein partners – Move to nucleus – Bind to enhancers – Act as activators 12-15
Glucocorticoid Receptors • DNA-binding domain with 2 zinc-containing modules • One module has most DNA-binding residues • Other module has the surface for protein- protein interaction to form dimers 12-16
Types II and III Nuclear Receptors • Type II nuclear receptors stay within the nucleus bound to target DNA sites • Without ligands the receptors repress gene activity • When receptors bind ligands, they activate transcription • Type III receptors are “orphan” whose ligands are not yet identified 12-17
Homeodomain-DNA Complex • Homeodomains contain DNA-binding motif functioning as helix-turn- helix motifs • A recognition helix fits into the DNA major groove and makes specific contacts there • N-terminal arm nestles in the adjacent minor groove 12-18
The bZIP and bHLH Domains • bZIP proteins dimerize through a leucine zipper – This puts the adjacent basic regions of each monomer in position to embrace DNA target like a pair of tongs • bHLH proteins dimerize through a helix-loop- helix motif – Allows basic parts of each long helix to grasp the DNA target site • bHLH and bHLH-ZIP domains bind to DNA in the same way, later have extra dimerization potential due to their leucine zippers 12-19
12.3 Independence of the Domains of Activators • DNA-binding and transcription-activating domains of activator proteins are independent modules • Making hybrid proteins with DNA-binding domain of one protein, transcription-activating domain of another • The hybrid protein still functions as an activator 12-20