? A 10 year old boy presented with signs and
symptoms and subsequent laboratory
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investigation reports suggestive of Acute
lymphoblastic leukaemia. Patient was
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receiving Methotrexate as a part of thistreatment. After several weeks of
chemotherapy with methotrexate, the boy
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showed signs of resistance to treatment. What
could be the most likely mechanisms to
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explain this resistance to methotrexate?2
Objective
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? What is Gene expression? Necessity of regulation of gene expression
? Different types of gene regulation
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? Gene regulation in prokaryotes
? Lac operon
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? Tryptophan operon? Gene regulation in Eukaryotes
? At transcription level
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? At translation
? Modification of DNA
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? DNA regulatory proteins3
? What is gene expression?
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? Formation of gene products
? RNA and Protein
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? Necessity of Gene Expression and regulation? Normal growth , development and functioning of
an organism
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? Production of gene product according to the
requirement of the body
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4Type of gene expression
? Constitutive
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? Occur all the time and at a relatively constant rate? Regulated
? Transcribed only when needed
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5
Regulation of gene expression
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? Positive regulation---- Enhancer/Activator? Negative regulation----Repressor/ Silencer
6
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Temporal responses to regulatory signalType A
observed in prokaryotes in response to sudden changes of
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the intracellular concentration of a nutrient
in higher organisms after exposure to inducers such as
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hormones, nutrients, or growth factors7
Type B
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occurs during development of an organism, when only the
transient appearance of a specific gene product is required
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8TypeC
Occurs during the development of differentiated function
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in a tissue or organ9
Stages of regulation of gene
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expression
? 1. Transcription
? 2. Post transcriptional modification
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? 3. Translation? 4. Post translational modification
? 5. RNA degradation
? 6. Protein degradation
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10Few specialized genetic and regulatory terms
Operon groups of genes sequentially arranged
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on the chromosome along with the regulatoryelements that determine their transcription
Polycistronic mRNA
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Constitutive /House keeping gene
Expressed at a constant level
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Inducible gene/Regulated egneexpressed only under certain condition
Gratuitous inducer: Acts as inducer but not as substrate
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An example is isopropylthiogalactoside (IPTG)
11
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Cis actingelements and
Trans acting
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molecules
12
Regulation of Prokaryotic Gene
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Expression
? Transcription is the primary site of regulation.
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13Role of Promoters
14
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Prokaryotic promoters share two regions of highlyconserved nucleotide sequence
Promoter: A regulatory region o DNA that serves to bind RNA polymerase
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I that in turn binds other substances that wil lead to initiation of transcription
15
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Adapted from Harper's BiochemistryOperon Model
Described by Jacob and Monod in 1961
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Hypothesis: Based on the regulation of lactose metabolism by the
intestinal bacterium E coli
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Led to the discovery of basic principles of Genetranscription activation and Repression
16
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Type of Operon? Catabolic Operon
? Regulates the gene expression whose product have a
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catabolic role
? e,g. Lac operon
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? Anabolic Operon? Genes of anabolic pathway such as synthesis of amino
acids are coordinately regulated
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? e,g. Tryptophan operon
17
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Lac Operon18
LacI gene encoding repressor
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CRE: cAMP response elementRegulatory region : Promoter : RNAP binds
Operator: Repressor binds
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Structural genes:
lac Z: Galactosidase
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lac Y: Permeaselac A:Thiogalactoside transacetylase
19
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? The operator locus is a region of double-stranded DNA that
exhibits a twofold rotational symmetry and an inverted
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palindrome (indicated by arrows about thedotted axis) in a region that is 21 bp long, as shown below:
The binding occurs mostly in the major groove
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The operator locus is between the promoter site and the
transcription initiation site of the lacZ gene
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2021
22
Lactose acts both as inducer and substrate
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23
Tryptophan Operon
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? Trp operon contains 5 structural genes? Trp operon is subject to negative control
? Trp facilitates the binding of the repressor to the
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operator
? Trp is a corepressor
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? Trp operon also regulated by attenuation? Transcription initiated but terminated wel before
completion
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Transcriptional attenuation occur in Prokaryotes
but not in Eukaryotes
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24Attenuation of transcription of the trp
operon when tryptophan is plentiful.
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25Coordination of transcription and
translation in prokaryotes
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? 1. Stringent response
? . 2. Regulatory ribosomal proteins
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26Regulation of
transcription by the
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stringent responseto amino acid
starvation
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27
Regulation of translation by an
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excess of ribosomal proteins.28
MCQ
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? Which of the following mutations is mostlikely to result in reduced expression of the lac
operon?
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? A. i? (no repressor protein made)
? B. Oc (operator cannot bind repressor protein)
? C. Cya? (no adenylyl cyclase made)
? D. Functionally impaired glucose transporter
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29
Regulation of Eukaryotic Gene
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Expression? Transcription is the primary site
of regulation
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? Role of Promoter
30
Schematic showing the transcription control regions in
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a hypothetical mRNA-producing eukaryotic gene
transcribed by RNA polymerase I
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31The eukaryotic basal transcription complex
32
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Stages of Regulation of EukaryoticGene Expression
? A. Coordinate regulation
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? 1. Galactose circuit
? Coordinated expressin mediated by Gal4
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? 2.Hormone response system? Intracellular receptors
? Cell surface receptors
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33Regulation of Galactose circuit in yeast
34
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Transcriptional regulation by intracellular steroid hormonereceptors.
GRE = glucocorticoid-response element
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(an example of a hormone response element); GR = glucocorticoid
receptor.
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35Transcriptional regulation by
receptors located in the cell
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membrane.
36
Regulation of Eukaryotic Gene
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Expression
? B. Regulation by co- and posttranscriptional
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processing of mRNA? 1. Splice-site choice/ Alternate splicing
? Tissue-specific protein products
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? tropomyosin (TM) regulates the functions of actin
in both muscle and nonmuscle cel s
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? 2. Alternative polyadenylation? 3. mRNA editing
37
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RNA editing of apo B in the intestine and generation of the
apo B-48 protein needed for chylomicron synthesis
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38? 4. mRNA stability
? A. IRON Metabolism
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? Regulation by mRNA degradation--Regulation of transferrinreceptor (TfR) synthesis
IRE = iron-responsive element; IRP = iron-responsive element binding protein.
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39
? Ferritin synthesis is regulated at the level of
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translation? Ferritin mRNA contains IRE at 5' UTR
? Binding with IRP prevents translation because it overlaps the
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translation initiation site? Iron availability is more: Fe binds to IRP-allows translation of
free mRNA of ferritin
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? Iron metabolism involves both mRNA stability and translation
level
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403. mRNA stability
--b. RNA interference (RNAi) by
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cleavage of target mRNA.miRNA
si RNA
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RNAi therapy:
Age-related macular degeneration (AMD) is triggered
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by overproduction of vascular endothelial growth factor(VEGF). The siRNA drug targets the mRNA of VEGF and
Promotes its degradation- injected in eyes.
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41
C. Regulation at Translation level
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Regulation of translation initiation in eukaryotes by phosphorylationof eIF-2. RER = rough endoplasmic reticulum.
42
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D. Regulation through modificationsto DNA
? 1. Access to DNA
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? 2. Amount of DNA:? 3. Arrangement of DNA
? 4. Mobile DNA elements
43
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1. Access to DNA
? Histone acetylation/deacetylation by the
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histone acetyltransferase and histonedeacetylase enzymes
? Extent of methylation of cytosine bases in CG
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rich regions (CpG islands) in the promoter
region by methyltransferases that use S-
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adenosyl - methionine as the methyl donor44
2. Amount of DNA:
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? Gene amplification : increase in copyNumber seen in response to particular
chemotherapeutic drugs such as methotrexate
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inhibitor of the enzyme dihydrofolate
reductase (DHFR),
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required for the synthesis of thymidinetriphosphate (TTP)
in the pyrimidine biosynthetic pathway
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? Resistance to the drug
45
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3. Arrangement of DNAV=Variable, D= Diversity, J= Joining
46
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4. Mobile DNA elements? Transposons comprise 50% 0f human genome
? Movement is mediated by transposase
? Movement can be
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? Direct
? Replicative
? Retrotransposon (90% of transposon)
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? Hemophilia, Duchenne Muscular dystrophy? Antibiotic resistance ?Exchange of Plasmids containing
Tn carrying antibiotic resistance genes
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47Summary
? Gene expression results in the production of a functional gene product
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(either RNA or protein
? Genes can be either constitutive or regulated
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? Regulation of gene expression occurs primarily at the level oftranscription in both prokaryotes and eukaryotes
? mediated through the binding of trans-acting proteins to cis-acting
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regulatory elements on the DNA
? In eukaryotes, regulation also occurs through modifications to the DNA,
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as well as through posttranscriptional and posttranslational events? In prokaryotes regulation is achieved through operons
? Operon: groups of genes sequentially arranged on the chromosome along
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with the regulatory elements that determine their transcription
? operon is induced by an isomer of lactose (al olactose)
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? The trp operon is also regulated by attenuation48
? Transcription of rRNA and tRNA is selectively inhibited in prokaryotes by
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the stringent response to amino acid starvation? Gene regulation is more complex in eukaryotes. Operons are not present,
but coordinate regulation of the transcription achieved through the
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binding of trans-acting proteins to cis-acting elements
? In multicellular organisms coordinated regulation, achieved through
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binding of hormone receptor?hormone complex to the DNA? Co and posttranscriptional regulation is also seen in eukaryotes
? Regulation at the translational level can be caused by the
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phosphorylation (inhibition) of eIF-2.
? Gene expression in eukaryotes is also influenced by availability of DNA to
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the transcriptional apparatus49
MCQ1
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? Which of the following is the basis for the
intestinespecific expression of apoprotein B-
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48?? A. DNA rearrangement and loss
? B. DNA transposition
? C. RNA alternative splicing
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? D. RNA editing? E. RNA interference
50
MCQ 2
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? Which of the following is best described as
being trans-acting?
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? A. CAP site? B. Operator
? C. Promoter
? D. Repressor
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51MCQ 3
? Which of the following is most likely to be true in
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hemochromatosis, a disease of iron accumulation in the
body?
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? A. The mRNA for the transferrin receptor (TfR) is stabilizedby the binding of IRPs to 3' stem-loop structures known as
IREs.
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? B. The mRNA for the TfR is not bound by IRPs, and is rapidly
degraded.
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? C. The mRNA for apoferritin is not bound by IRPs at its 5'stem-loop IRE, and is translated.
? D. The mRNA for apoferritin is bound by IRPs, and is not
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translated.
? E. Both B and C
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52MCQ 4
? After several weeks of chemotherapy with
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methotrexate, a cancer patient 's tumor begins to showsigns of resistance to treatment. Which of the following
mechanisms is most likely to explain this resistance to
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methotrexate?
? A. Deficiency of thymidylate synthase
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? B. Overproduction of xanthine oxidase.? C. Deficiency of PRPP synthase.
? D. Deficiency of thymidine kinase.
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? E. Overproduction of dihydrofolate reductase
53
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DNA regulatory protein? Proteins that regulate gene expression:
Repressor, enhancer, silencer
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? These proteins show several motifs which are
involved in these DNA protein interaction
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? Only a small part of the protein has directcontact with DNA
? Interactions are reversible and involve non
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covalent bonds
? Some commonly seen structural motifs
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54? Helix turn helix motif
? Zinc finger motif
? Leucine Zipper motif
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55Helix turn helix motif
E,g. Lambda repressor, CAP protein, Tryptophan repressor, Homeobox protein in mam5m6 als
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Zinc fingers are a series of repeated domains (two to nine) in whicheach is centered on a tetrahedral coordination with zinc.
Steroid thyroid receptor protein and calcitriol (vitamin D) receptor protein
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57
The leucine zipper motif
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58A schematic model of the DNA-binding domain of C/EBP
cfos.cjun or cjun.cjun
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59Transcription Factors That Contain DNA Binding Motifs
60
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61