bart mol biol 2009 - | siu school of medicinebbartholomew/bart mol biol 2009.pdf · 2009-08-27 ·...
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Contact informationBlaine BartholomewOffice: Neckers Bldg., Rm. 211 Phone: 453-6437 Email: [email protected]
Class noteshttp://www.siumed.edu/~bbartholomew/HbTxn.html
Other referencesPrinciples of Biochemistry by Lehninger 4th edition, pp 273-284, 938-943, 1082-1085, 1102-1104.
And for more in depth information (A) Thompson & Thompson, Genetics in Medicine, 1991 (B) Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second ed., 1998 (C)Connor & Ferguson-Smith, Medical Genetics, Fifth ed., 1996, (D) Jorde, Carey, Bamshad, & White, Medical Genetics, 2nd ed., 1999
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Developmental Expression of Human Globin Genes
from Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second Ed., p. 93, 1998
ζ2ε2 α2ε2ζ2γ2
α2γ2 α2β2 α2δ2
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Genomic Organization
Single copy: most common type with one copy/haploid
Duplicated genes: 2 or more copiese.g. histones
Gene families: closely related genes with similar but nonidenticalfunction
e.g. beta globin family
Pseudogenes: nonfunctional copies of functional genes that arise by gene duplication
e.g. b globin family
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Lodish, Fig. 10-3b
β Globin Gene Family
Functional Genes (expressed at different times during development)ε, Gγ, Aγ, δ, β
Nonfunctional Pseudogenesψβ2, ψβ1
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Human Globin Genes and HemoglobinsProduced During Development
β β
αα
Heme Developmental Period
Embryonic Fet al Adult
Hb Gower 1ζ2 ε2
Hb Gower 2α2 ε2
Hb Fα2 γ2
HbA2α2 δ2mi nor
HbAα2 β2
Hb Port landζ2 γ2
3’5’Chromosome 11 β-like genes
ε Gγ Aγ ψβ δ β
5’Chromosome 16 3’ α-like genes
ζ ζψ ψα ψα α2 α1
HbA
adapted from Thompson & Thompson Genetics in Medicine, p. 249, 1991
LCR
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Sickle cell diseases
• Sickle cell anemia– Caused by a single point mutation
• Sickle cell-hemoglobin C• Sickle cell beta-thalassemia
– Caused by mutations in the β globin gene locus
– Decreased or absent adult β globin synthesis
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One other version of these mutations
• Individuals with defective adult β globingenes can be phenotypically normally if …
• There are compensatory mutations that cause expression of fetal β-like genes after birth (Gγ and Aγ globin).
• This condition is called HPFH or hereditary persistence of fetal hemoglobin
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How is the expression of these genes regulated?The important underlying DNA
sequences and associated factors
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Eukaryotic & Prokaryotic TranscriptionRNA polymerases
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RNA PolymerasesA. E. coli RNA polymerase
1. core enzyme = ββ'(α)2 has catalytic activity but cannot recognize start site of transcription
~500,000 daltonsdimensions: 100 X 100 X 160 angstromsrequires Mg2+ for activityb' binds 2 Zn atoms
2. holoenzyme = core enzyme + sigma factor (s)carries out four functions:
(i) template binding(ii) RNA chain initiation(iii) chain elongation (iv) chain termination
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RNA PolymerasesB. Eukaryotic RNA polymerases (RNAP)
1. 3 nuclear RNA polymerases a. RNAP I- transcribes rRNA genesb. RNAP II - transcribes mRNA genesc. RNAP III - transcribes tRNA, 5S rRNA, and
other small RNA genesd. have 10-17 different subunits, large
multisubunit complexes are functionally similar to E. coli RNA polymerase
e. cannot bind to their respective promoters alone, but requires transcription factor for promoter specific recruitment
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RNA Polymerases
2. organelle specific RNA polymerasesmore prokaryotic-like
1. chloroplast2. mitochondria
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RNA Polymerases
3. RNAP IIa. core subunits - have sequence similarity to the
core subunits ofE. coli core RNA polymerase or subunits of other eukaryotic RNA polymerases
b. shared or common subunitssame subunits found in RNAP III and II or in RNAP I
and RNAP IIc. unique subunits - no similar homologs found
anywhere else
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How does RNA polymerase II find its correct binding site?
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Promoter StructureB. mRNA genes transcribed by RNAP II
1. TATA box element - located between -30 and -20 bps2. Initiator region or In: centered on the start site of transcription3. DPE: downstream promoter element4. Response elements (RE)
a. upstream of the TATA boxb. many different kinds - help respond to signalsc. multiple RE present - synergy
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How is transcription of particular genes get turned on in response to external stimuli such as stress
(heat, starvation, and so on), hormones and other small molecule effectors?
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Promoter Structure
B. mRNA genes transcribed by RNAP II5. enhancers
a. can be located at great distances (>1000 bps) from start site of transcription either from the 5' or 3' end of gene
b. stimulates transcription (~100 times)c. orientation independent
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Promoter Structure
B. mRNA genes transcribed by RNAP II5. enhancers
d. two models of how enhancers might work i. entry point of RNAP II by preventing
nucleosomes from binding or an altered DNA conformation that promotes RNAP II recognition
ii. transcription factors bound to enhancer will stimulate binding of RNAP II to promoter regions closer to the start site of transcription
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Transcription Factors
General versus promoter specific transcription factors.
Factors that are required for all mRNA genes and others that are required for only a small
subset of genes
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General Transcription Factors
Promoter Specific Transcription Factors
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General Transcription Factors
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Regulation of TranscriptionI. Basal vs. activated transcription for mRNA genes
A. General transcription factor (TF) vs. promoter-specific1. general TFs are required by all mRNA genes
a. an absolute requirementb. transcription can occur alone with these
factors and is by definition the basal level of transcription2. promoter-specific TFs are different for each gene3. the promoter-specific TFs are required for
maximal level of transcription or for activated transcription (induction)
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β − globin gene transcription factors
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Erythroid specific Transcription Factors
TAFII130, CBP/p300
346
basic leucinezipper
TGCTGA(C/G)TCANF-E2
Sp1, GATA-1,FOG, EKLF,
CBP/p300
GATA-likeC4 zinc
fingers
(A/T)GATA(A/GGATA-1
GATA-1, CBP/p300, TAFII130
Kruppel-like C2H2 zinc
finger
CACCCEKLF
INTERACTS WITH
CLASSMOTIFPROTEIN
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Regulation of TranscriptionII. Question of Activation
A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors
induction: heat shock, heavy metals, viral infection, growth factors, steroids
4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins
1. response elements shared2. example of homeodomains
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What is the difference between basal and activated transcription?
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What’s wrong with this picture?
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DNA is packaged into compact structures inside the cell
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Higher order structure – folding of chromatin fibers
Differing extents of compaction
Also this facilitates in DNA fitting inside the nucleus
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β− Globin Gene Cluster:Chromatin Structure
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Gaining Access to the DNA
Large multi-subunit protein complexes alter the state of
chromatin
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What is chromatin remodeling?
The process of making DNA more or less accessible in the eukaryotic genome using
a series of specialized proteins
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Accessibility is Key Regulatory Step
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ATP-dependent chromatin remodeling machinesDifferent classes of chromatin
remodeling complexes
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Different classes of ATP-dependent chromatin remodeling complexes
A. SWI/SNF or SWI-SNF like complexes– required for activation of a small subset of
genes– found in humans, flies, and yeast– at least two different forms of the complex in
yeast or humans– in yeast it has 11 or 17 subunits– total complex size is 1.5 -2 Megadaltons
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SWI/SNF or SWI/SNF-like Complexes Are Found in All Eukaryotes
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Activities associated with ATP-dependent chromatin remodeling• Translate nucleosomes along DNA
– Change translational position (general)– Nucleosome spacing (ISWI)
• Displace or dissemble nucleosomes (SWI/SNF and RSC)
– Octamer transfer– Loss of H2A/H2B dimer
• Histone exchange– Example of H2A exchanged for H2AZ by SWR1
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Bulge Diffusion Model for chromatin remodeling
• Initiates from outside the Nucleosome• Initially breaks contacts at edge of Nucleosome•These two point are also true for other model(s)
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DNA can be cut by nucleases (LCR)
Site 3 has SWI/SNF bound at this region
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Chromatin Remodeling • ATP-dependent chromatin remodeling
– Moves nucleosomes along DNA– Involved in displacing nucleosomes from DNA– Exchanges parts of the nucleosome
• Covalent modification of histone proteins– ACETYLATION– METHYLATION– PHOSPHORYLATION– UBIQUITINATION– Poly ADP RIBOSYLATION– SUMOLYATION
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Sites of Histone Modification
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Histone acetyltransferases (HATs)
A. cytoplasmic HATs (Type B)i. acetylate newly synthesized H3 and H4 histones
before depositing on to DNAii. after binding to DNA the acetyl groups are
removed
B. nuclear HATs (Type A)i. have been found to be required for gene
activationii. part of TFIID has HAT activity (TAFII250)iii. these HAT proteins are usually large multi-
subunit complexes
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Mapping sites of histone modifications
• Have developed a technique called ChIP(Chromatin Immunoprecipitation)
• Identify the genomic location where different histone modifications reside
• Is dependent on antibodies that recognize the particular modification
• Can be used to examine the entire genome
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Pattern of histone acetylation on the β globin gene
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Chromatin
Euchromatin - dispersed (acetylated) chromatin, gene rich regions of the genome
Heterochromatin - chromatin is condensed (deacetylated), gene poor regions of the genome
human genome 3 x 109 bp with an estimated 20-25,000 genes only 2- 4% of the DNA codes for proteins
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Histone acetylation generally associated with gene activation
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Summary of regulatory elements
What is an insulator element (which is similar to what is also called a boundary element)?
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Insulators: Two models
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Nuclear Matrix or Scaffold
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DNA Loops/ Domains
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Chromosome Territories
Individual chromosomes occupy distinct regions in the nucleus
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Chromosome Territories
• Gene rich chromosomal regions– Tend to be oriented toward the nuclear
interior• Gene poor chromosomal regions
– Tend to be associated with the with nuclear periphery
• Dynamic reorganization
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