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Gene expression
• Review sessions wt Jackie: 3-5PM Thursdays, room 8 Illick
• Previous: www.esf.edu/efb/powell/Gen_L8_3.pdf www.esf.edu/efb/powell/Gen_L8_6.pdf
www.esf.edu/efb/powell/Gen_L9_3.pdfwww.esf.edu/efb/powell/Gen_L9_6.pdf
www.esf.edu/efb/powell/Gen_L10_3.pdfwww.esf.edu/efb/powell/Gen_L10_6.pdf
Next Tuesday:www.esf.edu/efb/powell/Gen_L11_3.pdfwww.esf.edu/efb/powell/Gen_L11_6.pdf
Lac operon in E. coli
• What if glucose and lactose are present?– Note: takes less resources to use
glucose• Lac operon is not turned on• Catabolite Repression
– A product of glucose catabolism inhibitsthe formation cAMP (cyclic adenosinemonophosphate)
– Increase glucose = decrease cAMP– Decrease glucose = increase cAMP
Lac operon in E. coli
• How does cAMP effect the Lac operon?• cAMP binds to a protein called CAP (catabolite
activator protein)• cAMP/CAP increases binding of RNA polymerase to
the Lac operon’s promoter, therefore increasesexpression
• cAMP/CAP - example of positive control (required foractivation)
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Catabolite repression of Lac operon(Fig 17-8 in your textbook)
Glucose present, no lactose
No lac mRNA
(Low cAMP)
Glucose present, lactose present
little lac mRNA transcribed
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No glucose present, lactose present
Abundant lac mRNA
Comparison of negative & positivecontrol
• negative control– Repressor protein - prevents expression
• Positive control– Activator protein - enhances expression
Inducible vs. repressible systems
• Lac operon is an example of aninducible system (gene is normally off)
• Repressible systems work in a similarway, but their expression is normally on(example: trp operon in textbook)
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Gene regulation
• Simple to complex• Human genes average 5 cis-acting
elements which bind as many trans-acting regulatory proteins. Some geneshave up to 20.
Regulatory cascades
Gene Aon off
Regulatory cascades
Gene Aon off
Gene(s)
gene(s)
gene(s)
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Regulatory cascades
Gene Aon off
gene(s)
gene(s)
gene(s)
Developmental Gene Regulation
• Governed by regulatory cascades• Master switches
– Genes that commit development down onepath or the other
– Example: sex determination
Gene regulation in Eukaryotes
• So far looked at model system– Prokaryotic lac operon
• Eukaryotic systems similar, but much morecomplex
• Eukaryotes have 3 RNA polymerases– RNA polymerase II transcribes all mRNA
• RNA polymerase must bind to the promoterfor transcription to initiate– Many proteins that assist in binding
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Eukaryotic promoters
• Have additional cis-acting elements• Enhancers - enhance RNA pol. binding• Silencers - inhibit RNA pol. binding• Both act like a “volume switch” turning
expression up or down (not just on or off)• Often a given enhancer or silencer activity is
tissue specific• These elements can be located close or far,
upstream or downstream from core promoter
Core promoter & proximal elements
• Core promoter - transcription start site, 30bases upstream to a TATA box– Where the RNA polymerase binds to DNA
• Promoter proximal elements - 100-200 basesupstream from transcription start site– CCAAT box– GC-rich region
• These are the minimal promoter sequencesneeded for transcription initiation
Core promoter & proximal elements
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Complexity of a eukaryotic promoter
Enhancers help control physiology &development
• In eukaryotic organisms, cells in onepart of the body can control geneexpression in cells of other parts of thebody (global gene control)
• Example: the human endocrine system– Produce steroid hormones– Genes (cell#1) > enzymes > hormones >
travel through blood > receptor protein(cell#2) > binds to enhancer on gene
peptide hormones
Endocrine organs:
Sex determinationFight/flightMetabolic rate Sugar regulationGrowthEtc.
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Hormone responsive enhancers(inducible system, positive control)
Development & sex determination
• Opportunities forgene regulation
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Development of male vs. female
Sex determination
Fruit fly Human
XX
XY
female female
male maleAppear the same
aneuploids
XXY
XO
female male
femalemale
Shows sex isdetermined bydifferentmechanisms
Review of gene expression
pre-mRNA
transcription by RNA polymerase
RNA processingmessenger RNA (mRNA)
translation
polypeptide (protein) post-translationalprocessing
DNA
genetranscriptional unitPromoter
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Drosophila (fruit fly)
• Basis of sex determination: X:A ratio of a gene– X = X chromosome copy– A = autosome (non-sex chromosome) copy
• Female: X:A ratio of one• Male: X:A ratio of 0.5• How can the developing embryo count the
chromosome copies of genes?• Model: what is thought to happen from current
evidence
Drosophila• 2 genes encode for sex determining transcription
factors (TF)– Transcription factor helps to bind RNA polymerase to
DNA• Autosomal copy - has no functional DNA binding
domain• X chromosome copy - has a DNA binding domain• These TFs will form dimers in the cell
– Dimers - combination of two polypeptides– Must have two functional polypeptides to bind to DNA
transcription factors (TF)Female: 1:1 ratio Male: 1:2 ratio
X chromosomes X chromosomesautosomes autosomes
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transcription factors (TF)Female: 1:1 ratio Male: 1:2 ratio
X chromosomes X chromosomesautosomes autosomes
active Inactive
transcription factors (TF)Female: 1:1 ratio Male: 1:2 ratio
X chromosomes X chromosomesautosomes autosomes
active Inactive
Much more inactive than active
Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
intron
Two promotersLate & Early
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Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxlprotein
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Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxlprotein
Feedback to maintain splicingwhen late promoter is on.
Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxlprotein
Feedback to maintain splicingwhen late promoter is on.
Not enough active Tfsto turn on earlypromoter
Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxlprotein
Feedback to maintain splicingwhen late promoter is on.
Not enough active Tfsto turn on earlypromoter
Unspliced mRNA
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Sxl gene (female specific splicing)
Female: Male:
PEPLPEPL
Many active Tfsturn on early promoter
Splicing ofmRNA
Sxlprotein
Feedback to maintain splicingwhen late promoter is on.
Not enough active Tfsto turn on earlypromoter
Unspliced mRNA
UGA stop codon preventstranslation of Sxl protein
Drosophila
• Therefore XXY = female, X0 = male– XXY ratio is 1:1– X0 ratio is 1:2 (or 0.5)
Sxl master control switch
• Controls its own splicing and also starts acascade of gene expression
• tra gene - transformer– Female (active Sxl) - forms active tra RNA binding
protein– Male (no Sxl) - tra inactive
• Tra controls the RNA splicing of the dsx gene– Two forms of protein product
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dsx gene (negative control, repressible system)
• Female dsx protein– Represses male-specific structural genes
(many)• Male dsx protein
– Represses female-specific structural genes(many)
CascadeFemale: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
CascadeFemale: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
tra (on) tra (off)
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CascadeFemale: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
tra (on) tra (off)
dsx mRNAfemale splice
dsx mRNAmale splice
CascadeFemale: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
tra (on) tra (off)
dsx mRNAfemale splice
dsx mRNAmale splice
dsx-F protein dsx-M protein
CascadeFemale: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
tra (on) tra (off)
dsx mRNAfemale splice
dsx mRNAmale splice
dsx-F protein dsx-M proteinRepression ofmale-specific genes
Repression offemale-specific genes
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Drosophila• Null mutation of Sxl?• Male• Null mutation of Tra?• Male• Therefore, male is default pathway• Null mutation of dsx• Characteristics of both male and female
– No suppression of genes
Female: X:A = 1 Male: X:A = 0.5
Sxl (on) Sxl (off)
tra (on) tra (off)
dsx mRNAfemale splice
dsx mRNAmale splice
dsx-F protein dsx-M protein
Repression ofmale-specific genes
Repression offemale-specific genes
Sex determination in mammals
Comparison: Drosophila MammalsX:A ratio
Sex determination in mammals
Comparison: Drosophila MammalsX:A ratio Y chromosome
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Sex determination in mammals
Comparison: Drosophila MammalsX:A ratio Y chromosome
autonomous non-autonomous every cell global
Sex determination in mammals
Comparison: Drosophila MammalsX:A ratio Y chromosomeautonomous non-autonomousevery cell global
mosaics: male and female male or female
Sex determination in mammals
Comparison: Drosophila MammalsX:A ratio Y chromosomeautonomous non-autonomousevery cell global
mosaics: male and female male or female
Why? - secondary sexual phenotypes are driven by the presence or absence of the testes
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Human development
• Gonads form within first two months fromconception
• If Y chromosome is present, then testes form• If no Y, then ovaries form• testes secrete testosterone (steroid hormone)• Testosterone binds to androgen-receptors in
all the cells of the body (global)• These act as transcription factors which
enhance male specific gene expression
Human development
• If no testosterone, androgen-receptors are notactivated, therefore female genes are expressed bydefault
• What would happen if a null mutation occurred in theandrogen-receptor gene?
• Testicular feminization (Tfm) syndrome– XY female– Female secondary characteristics, but has testes
instead of ovaries (demo. of global affect)• Several types of mutations that effect sex
determination
(Caster)
Human development
• Testosterone - developmentally specific for sometraits– developmental timing is important
• Female + testosterone ≠ male– Facial hair, increased muscle mass, more
aggressive• What drives the gonads to form testes?• SRY gene (old name TDF: testes determining factor)
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SRY gene
• Sex-determining region of Y chromosome• Identified by studying sex reversal
– XX males• Caused by translocation of portion of Y
chromosome onto an X chromosome• Proved SRY gene is the sex-determining
master switch by cloning into XX mice
SRY gene
PCR markers (Zfy-1 = Y chromosome)