circadian rhythm -...
TRANSCRIPT
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 1
Circadian Rhythm
- freerunning, sustainable oscillation with an approximately 24 hour period
- entrainment to daily environmental changes
- universally associated with light and temperature perception
- period length of rhythm is compensated against changes in temperature or metabolic state
- clock operates within defined physiological limits
0 12 0 12 24
Sound recorded from fourCastor canadensis
March 18 - April 22, 1971115°03'W, 51°02'N
.
EUKARYOTAFUNGI
Sponges
ANIMALIA
CiliatesBrown AlgaeDiatoms Protista
Giardia lamblia
Vairimorpha necatrix
Trichomonas foetus
Physarum polycephalumEuglenoids
KinetoplastidsBodonids
Amoebamastigote
Entamoebae invadens
Escherichia coli
CyanobacteriaPlant Chloroplasts Mycoplasma
capricolum
Pseudomonas testosteroniPlant Mitochondria
Agrobacteriumtumefaciens
Dinoflagellates
Dictyostelium discoideumRed Algae
EUBACTERIA
ARCHAEBACTERIA
Mammals-mouse, human
Neurospora
ArabidopsisChlamydomonas
ANIMALIAGonyaulax
Ciliates
TetrahymenaParamecium
Brown Algae
Dinoflagellates
FUNGI
PLANTAE
Insects - Antheraea,Drosophila
Diatoms
Synechococcus
PLANTAE
Circadian Systems in the Universal Tree of Life
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 2
WHITE COLLAR-1 & WC-2 in NeurosporaCLK & CYC in Drosophila
CLOCK & BMAL1 in mammals
Negative elements in circadian loops:
FREQUENCY in Neurospora
PERIOD and TIMELESS in Drosophila
PER1, 2, and 3 , CRY1 and CRY2 in mammals
Positive elements in circadian loops:
"Positive Element(s)"
"Negative Element(s)"
output - clock controlled genes
clock gene(s)
rhythmicbiochemistry andphysiology
promoting expression
blocking activity
Common Themes and Elements in Eukaryotic Circadian Feedback Loops
WHITE COLLAR-1 & WC-2 in NeurosporaCLK & CYC in DrosophilaCLOCK & BMAL1 in mammals
Negative elements in eukaryotic circadian loops:
FREQUENCYin NeurosporaPERIOD and TIMELESS in DrosophilaPER1, PER2, PER3 , CRY2, and CRY2in mammals
Positive elements in eukaryotic circadian loops:
output - clock controlled genes rhythmicbiochemistry,physiology,or behavior
Common themes:Transcription/translation based feedback loops, with transcription factors as positive elements
and proteins that block the activity of these factors as negative elements, are central.Rhythmic expression is seen in transcripts and proteins corresponding to the "negative elements.""Positive elements" are heterodimers comprised of two proteins interacting via PAS domains.
Of proteins comprising the heterodimer, one is constitutive and one is rhythmically expressed.Extended sequence similarity links some of the positive elements among all of the systems.At least one of the "negative elements" has a dual role in the cycle, performing both the negative function
in feedback and also a positive function in promoting the expression of one of the heterodimer partners.
clock gene(s)"Negative Element(s)"
blocking activity"Positive Element(s)"
promoting expression
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 3
Molecular components of the Neurospora clock
frq
wc-2
wc-1vvd
ccg’s
WC-1
FRQ
FRQ
FRQkinases:CK-1αα, Ck-1ββ,CAMK-1, CKII
turnover
FRQ
FRQ
P
P
P
P
P P
P
-
+
+
+
+
FRQfrq
nightnightday
WC-2
WC-1
WC-2WC-1
WC-2WC-1
nucleus
•WC-1 and WC-2 act positively on the production of frq transcript.
•FRQ acts negatively on the production of its own transcript. (negative feedback loop)
•FRQ acts positively on the production of wc-2 transcript and WC-1. (positive feedback loops)
•FRQ becomes phosphorylated which leads to its turnover
-
•The frq/FRQ oscillations are essential for the generation of all circadian rhythms in Neurospora.
•Changes in frq/FRQ levels are directly translated into changes in the circadian rhythms in Neurospora.
Cry1
Cry2
PER1
LIGHT
Per1Per2
ccg's
Clock-controlled processes
SCN neuronal activity rhythmsneuropeptide release
overt rhythms in the organism
Per3
PER3PER2
phosphorylation &
turnover ?
complexformation
Nucleus
PERyPERx::(andothers)
BCCBMAL1::CLOCK
Cell
mammalian neuron
Cry1
Cry2
Cry1ccg's
Clock-controlled processes:stress responsesdevelopmental regulationintermediary metabolism
LIGHT
frqkinase
turnover
Nucleus
FRQPP
processing
Cell
Neurospora
wc-1
vvd
FRQWCC
WCC
translation
WCC
wc-2
PP
FRQproteins
Cry2
bmal
two eukaryotic circadian oscillators
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 4
Circadian rhythm
•Biological rhythm with a period of ~24 hours•Endogenous and self-sustaining under constant environmental conditions•Temperature compensated within the physiological range of the organism •Environmental signals (light and temperature changes) can entrain or reset the rhythm•Organized at the cellular level
Van D. GoochUniversity of Minnesota
A Circadian Rhythm in Development on Racetubes
PRC movie
ENTRAINMENTLight or temperature treatment results in changes in the timing of conidial banding.
Van D. GoochUniversity of Minnesota
QuickTime™ and aSorenson Video decompressorare needed to see this picture.
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 5
WC-1
Time
FRQproteins
ccg's
Clock-controlled processes:stress responsesdevelopmental regulationintermediary metabolism
caseinkinase II
turnover
NucleusPP
FRQPP
processing
Cell
wc-1
vvd
FRQWCC
WCC
translation
WCC
wc-2
Light
frq
Molecular Components of theNeurospora Clock
FRQFRQfrqfrq
frq +
mRNA
203
118FRQ
11 18 25 47DD 33 4015 22 29 36 43 51 1815 hoursCT 0 8 16 16 4 84 12 20 0 4 12 208
frq + frq10
0
5
10
15
20
0
5
10
15
20
11 15 18 22 25 29 33 36 40 43 47 51
Hours in Constant Darkness
FRQfrq+
mRNArRNA
Rhythms in frq mRNA and FRQ protein
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 6
205
116
0 3 7 12 15 19 0 3 7 12 15 19CTIP + λλPPaseExtract
FRQ is Phosphorylated in a Time-of-Day-Specific Manner
High temp
Low temp
.
frq
513I
513D
64200.2
0.4
0.6
0.8
1.0
1.2
bd513I513D
Hrs after addition of CHX
Rel
ativ
e F
RQ
leve
ls
Phosphorylation site at aa 513 is important for FRQ degradation
+
PERIOD LENGTH
ARR
35 hr
22 hrYL 5/99
0 1 3 5 0 1 3 5 0 1 3 5control 513I 513D
Hrs:
FRQ
SER->ILE SER->ASP
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 7
.
T ot al L ys at e Nu cl ear
p C L 1 1 p C L 1 1
AUG#1AUG#3
N-terminal NLS frq(pCL11)
p C L 1 1 p C L 1 1
A.
Re-insertion of the NLS restores nuclearlocalization to polypeptide bearing it.
NLSfrq +
1st AUG3rd AUG
frq ORF
frq ORF1st AUG
3rd AUG
frq
+
frq
∆∆ NLS
frq
(nu
ll)10
frq
+
frq
∆∆ NLS
Re-insertion of the NLS restores FRQ function.C.
NLS
Nu cl ear
∆∆ AU
G#!
∆∆ AU
G#3
frq
+
frq
+
phosphatase t reated
T ot al L ys at e
B.
frq +/25°C
pCL11/25°C
pCL11/27°C
pCL11/22°C
frq ∆∆NLS/ 25°C
FRQ acts in the
nucleus
Molecular Components within the Neurospora Circadian Oscillator
FRQproteins ccg's
Clock-controlled processes:stress responsesdevelopmental regulationintermediary metabolism
LIGHT
frqCK 2
turnover
Nucleus
FRQPP
processing
Cell
wc-1
vvdWCC
translation
WCC
wc-2
FRQPP
WCC
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 8
WC-1 and WC-2 are par t of theNeurospora clock
5 15304560
-light +light
2 ' li ght pulse
5 153045 60 5 15304560
5 15304560
frq
frq WTwc-1
DD LP DD LP DD LP
ER33 ∆∆wc-2 WTfrq
frq0 2 6 1014 18 2226
FRQ
2 6 10 14 18 2226 30 34 38 42 46 50 54 58
LL, 4°C -> DD, 25°C
WT4°
frq
0 2 6 101418 2226
FRQ
2 6 10 14 18 22 26 30 34 38 42 46 50 54 58
wc-1frq
0 2 6 1014 18 2226
FRQ
2 6 10 14 18 22 26 30 34 3842 46 50 54 58
wc-2[ER33]
4°
4°dark
loss of function
wc-2
White Collar-1 (WC-1) and White Collar-2 (WC-2)
•WC-1 and WC-2 are required for the light induction of frq transcript.
•WC-1 and WC-2 heterodimerize via their PAS domains.
•WC-1 and WC-2 contain zinc-finger (Zn) DNA binding domains and activation domains (AD).
WC-2PAS ZnAD 530 aa
WC-1LOV PAS PAS ZnAD AD 1167aa
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 9
.
Anti-WC2 co-immunoprecipitatesFRQ and WC1
FRQ
WC2
WC1
I PIt o t a l e x t r a c t
DD26
I PI
DD14
FRQ
WC2
WC1
A Partially Functional Allele of wc-2 Displays Altered Period Length and
Temperature Compensation
WTER24
Temperature (�
C)
Per
iod
len
gth
(h
ou
rs)
20
22
24
26
28
30
20 22 24 26 28 30
wild type
wc-2ER24
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 10
WC-1
FRQ
WC1 vs. FRQ in frq+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
4 12 20 28 36 44 52 60 68
WC1 vs. FRQ in frq 7
0
5
10
15
20
25
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
4 12 20 28 36 44 52 60 68
W C1
FRQ
Hours in constant darkness Hours in constant darkness
Protein cycling in frq+ Protein cycling in frq7WC-1
FRQ
Levels of WC-1 and FRQ cycle in Antiphase to One Another
..
ATGqa-2promoter
FRQ ORF
+
=
quinic
acid
glucose( )
( )
VII R
qa-1S qa-1F(activator)(repressor)
Indu cible FRQ Protein Production
qa-1Fqa-1S
FRQ
protein
3’ UTR
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 11
FRQ promotes the Synthesis of WC-1 through a Post-transcriptional
Mechanism
+frq10::qaFRQ
+frq+
ααWC-1QA
ααFRQ
--
1 2 3 1 2 3 1 2 3 1 2 3
QAfrq+
wc-1EtBr stainedmembrane
- -+ +frq10::qaFRQ
wc-1 RNArRNA
- + +
frq+
-
50
100
frq10::qaFRQ
- + +frq+-
frq10::qaFRQ
relativeamountofprotein
0 4 8 12 16 20 0 4 8 12 16 20
QA pulse cont r ol
ααWC-1
ααFRQ
hr oftreatment
16
8
00 8 16
Time (hr)
relativeamount
ofprotein
ααFRQ(longer exp.)
50
100
50
100
WC-1
Time
FRQproteins
ccg's
Clock-controlled processes:stress responsesdevelopmental regulationintermediary metabolism
caseinkinase II
turnover
NucleusPP
FRQPP
processing
Cell
wc-1
vvd
FRQWCC
WCC
translation
WCC
wc-2
Light
frq
Molecular Components of theNeurospora Clock
FRQFRQfrqfrq
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 12
42342618100
2
4
6
8
10
1221°C FRQ28°C FRQ
Time in DD (hr)
Leve
l of F
RQ
42342618100
2
4
6
8
10
12
Time in DD(hr)
Leve
l of f
rq
FRQ
frq
1318 24 29 3440
21 Co
1318 24 29 3440
28 Co
rRNA
amidoblack
Hrs:
FRQ Oscillates around a Higher Levelat Higher Temperatures
21°C frq28°C frq
CT 0 12dawn dusk
CTSTEP UP: All points on the low tempcurve are low compared to the high tempcurve, so the clock is reset to the timecorresponding to the low point in FRQ - dawn.
CT 0 12dawn dusk
CTStep Down: All points on the high tempcurve are high compared to the low temp curve,so the clock is reset to the time correspondingto the high point in FRQ - late day.
time of day
amo
un
t o
f cl
ock
co
mp
on
ent
Clock Resetting by Temperature Steps
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 13
.
ribosomal RNA
10'
20'
30'
60'
120'
frq RNA
seconds of light ( 25 lux )
0'
Light-Induced Clock Resetting Increases in Proportion to Light Induction of frq
1
5
1 0
level of frq RNA vs. light
seconds of light (25 lux)
0 10 20 30 60 120
frq RNA
ribosomal RNA
0
2
4
PhaseAdvance
(hrs)
0 4 8 12 16 2018
AdvanceDelay
0
-6
6
12
frq
mR
NA
leve
l
Subjective DAY NIGHTNIGHT ligh
t-in
duce
d cl
ock
rese
ttin
g
hrs in dark- light
frq
+ light28 32 36 40 44 48 28 32 36 40 44 48
frqRNA
Advance
Delay
Resetting the Neurospora Clock with Light
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 14
Digoxygenin staining of SCN +/- light
Shigeyoshi et al,Cell, 1997
frq
frq
How are light signals perceived and transduced to the clock?
•frq transcript is rapidly and highly induced in response to light.
nightnightday
?
?
?
+WC-2WC-1
•WC-1 and WC-2 are required for the light induction of frq transcript.
nightnightday
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 15
The frq promoter contains two Light Response Elements (LRE) necessary and sufficient for light induction
� distal
& �
proxwt
� distal
� prox
frq RNA*
*
* = light
= dark
proximalLRE
distalLRE
ClaI MscI SnaBI BglII MscI +1
~1200 bp ~200 bp
ααW
C-1
ααW
C-2
ααF
RQ
wc-
1ER
45
wc-
2KO
frq
KO
self non-self mut.self
1 4 532 6 7 8 10 119 12 13 14 1615 17
The LREs are bound by a WC-1/WC-2 containing complex
WC-2WC-1
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 16
The distal and proximal LREs are bound by distinct complexes in the light and dark
Light
Dark
Light
Dark
proximal LRE distal LRE
growth conditions
What causes the change in mobility of the WCC/LRE complex in the light?
•phosphorylation of WC-1/WC-2
•conformational change of WC-1/WC-2
•binding of additional factor(s)
•combination of the above
•other?
?
PPP
P
P PP
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 17
VVD
PYP
W C-2
W C-1
PAS domain
DNA binding domain
activation domain
His kinasedomain
chromophore
100 aa
Real and Potential Neurospora Photoreceptors
Response regulator-receiver
GAF+ chromophore
PHOT1
PHYA
PHY-2
PHY-1
CRY
CRY
known photoreceptors from other systems
LOV domain
NOP-1
Nuclear Protein Extracts are Photoreactive
growth conditions D DL L
in vitrolight treatment
L L
Protein extractions, binding reactions and gel running performed in the dark (red lighting).
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 18
Is the in vitro light-induced change in mobili ty of the WCC/LRE complex occurr ing at biologically relevant…
•amounts of light (ill uminance)?
•quali ties of light (wavelength)?
Light (umol photons/m2)
0.0
0.2
0.4
0.6
0.8
1.0
0 10 20 30 40 60 180
300
900
600
1800
1200
2700
1800
0
9000
3600
Rel
ativ
e B
and
Inte
nsi
ty
The in vitro light-induced WCC/LRE mobil ity change occurs at biologically relevant light intensities.
in vivo threshhold
1
5
10
In vivo light responses
0 8 16 24 48 96
frqmRNA
0
2
4 PhaseAdvance
(hrs)
Light (umol photons/m2)
Crosthwaite et al, Cell, 1995
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 19
Wavelength (nm)0 410 455 540430 475
0.0
0.2
0.4
0.6
0.8
1.0
400 450 500 550
Rel
ativ
e B
and
Inte
nsi
ty
1012
1013
Ban
din
g In
hib
ition
(1/erg s
-1cm-2)
Sargent and Briggs Plant Phys. 1967
The in vitro light-induced WCC/LRE mobility change occurs at biologically relevant wavelengths.
Slower Complex
dark light
Faster Complex
D L
WC-1 and WC-2 generated in vitro can bind to the LREs as both the faster and slower migrating complexes
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 20
Slower Complex
dark dark lightlight
- FAD +FAD
Faster Complex
WC-1 +FADWC-1
WC-2
D L
D LD LD L
FAD imparts light regulation to the WC-1/WC-2 complex
FAD
How are light signals perceived and transduced to the clock?
• frq transcript is rapidly and highly induced in response to light.
• The frq promoter contains two cis-acting Light Response Element (LREs).
• The LREs are bound by a WC-1/WC-2 heterodimeric complex in the dark.
• Absorption of light by a FAD chromophore bound by WC-1 triggers the multimerization of the WCs presumptively allowing the multiple WC activation domains to act.
proximalLRE
distalLRE
+1
frq promoter
FAD FAD
Therefore, WC-1 is a circadian blue light photoreceptor.
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 21
frq
wc-2
wc-1vvd
ccg’s
WC-1
FRQ
FRQ
FRQkinases:CK-1αα, Ck-1ββ,CAMK-1, CKII
turnover
FRQ
FRQ
P
P
P
P
P P
P
-
+
+
+
+
FRQfrq
nightnightday
WC-2
WC-1
WC-2WC-1
WC-2WC-1
nucleus
•WC-1 and WC-2 act positively on the production of frq transcript in the dark.
-
0 2412 36
The distal LRE is necessary for overt and molecular rhythmicity
Light Darkwt
� distal
wt
4 25oC
� distal
0 2412 36
frq RNA FRQ protein
� distal
wthours in dark hours in dark
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 22
The distal LRE is sufficient to drive rhythmic transcriptionThe distal LRE is also a circadian element.
6 12 18 24
endogenousfrq
vectorhph
reporter
distal LRE
6 12 18 24hours in dark
*
hours in dark
proximal LRE
hp
hTATA
hph{LRE}
The distal LRE is rhythmically bound by the WCC
12 3630 422418DDCT 23 2216 3115
12 18 24 30 36 42DD
FRQBinding
CT 23 2216 3115
WC-1
WC-2
FRQ
12 3630 422418 DDCT23 2216 3115
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 23
•FRQ negatively regulates its own transcript.
•FRQ physically interacts with WC-1 and WC-2.
•Oscillations in FRQ levels are phased appropriately and suggest an attractive means of generating oscillations in WCC binding.
Does FRQ regulate WCC binding to the distal LRE?
WC-2
WC-1
FRQ
Gel Shift
Hrs Induction
Strain
0 04 421
wtQA::FRQ
0 1 2 4
wtQA::FRQ
0 4 Hrs Induction
Strain
Bin
din
g
FR
Q
0
1
2
3
4
5
6
7
0
4
8
12
16
20
24
Increases in FRQ cause a decrease in WCC binding
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 24
Dawn ~CT0/24•FRQ increasing•WCC binding decreasing•frq transcription high
Midnight ~CT18-22•FRQ precipitously turns over and is low•WCC binding increasing or maximal•frq low, but increasing
Midday ~CT6-10•FRQ increasing to maximum•WCC binding decreasing to minimum•frq decreasing
Dusk ~CT12•FRQ decreasing, becoming phosphorylated•WCC binding low•frq low
FRQfrq
nightday
binding
Model of the Neurospora Clock (in constant darkness)
AcknowledgementsClock Genes and Clock Mechanism
Hildur ColotSue Crosthwaite
Mi ShiDeana Denault
Allan Froehlich
Kwangwon Lee
Antonio Pregueiro
Jill Wahleithner
Hitoshi Okamura
Giles Duffield
Bill SchwartzKobe Univ.
Mouse
U. Mass. Worcester
Neurospora
Han Cho
Light RegulationChristian Heintzen
Yi Liu
Allan Froehlich
Clock-Controlled Genes
Bruce Roe
Univ. OklahomaBernd Meurers
RW Johnson PRI
Mari ShinoharaMinou Nowrousian
Jennifer LorosJay Dunlap
Carol Ringelberg
Carsten Schwerdtfeger
Arun Mehra
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 25
23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1Fraction #
Rel
ativ
e P
rote
in
WC-1/lightWC-2/light
WC-2/darkWC-1/dark
WC-1
WC-2
dark
** *
22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1TLFraction #
+light WC-1
WC-2
ΒΒ-amylase200kDa
*
BSA66kDa
* *
thyroglobin669kDa
23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1Fraction #
TL
Light-Induced Shift of WC-1/WC-2 into a Higher Molecular Weight Complex
?
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 26
Dawn•FRQ increasing•WCC binding high•frq transcription high
Midnight•FRQ decreasing•WCC binding increasing•frq low, but increasing
Midday•FRQ high•WCC binding high•frq transcription high
Dusk•FRQ high, but decreasing•WCC binding decreasing•frq decreasing
FRQfrq
nightday
binding
Model of the Neurospora Clock (under light/ dark cycles)
Deletion of the proximal LRE results in a light-specific phase delay
wt
4 25oC
� prox
wt
Light Dark
� prox
~12 hr delay
� prox
wthours in dark
frq RNA
0 12 24 36
FRQ protein
0 12 24 36hours in dark
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 27
WC-2WC-1
Consensus sequence for WC-1/WC-2 binding
distal LRE
CGTCCTGATGCCGCTGCAAGACCGATGACGCTGCAAAATTGAGATCTA**** ****
CGCAGAGGACCCTGAACTTTTCGATCCGCTCGATCCCCTGGAA
proximal LRE
**** ****
proximal
distal
CGATCCGATCTGATGCGATGCGATACCGATAATACGCTal-3
CGCT
CCGCTACGCTCCGCA
CCCT
CGAT_____CGCTconsensus
The WC-1 LOV domain shares homology with LOV domains found in blue light photoreceptors
LOV WC-1PAS PAS ZnAD AD
chromophore
Crosson and Moffat, 2001
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 28
= flavin
Flavin adenine dinucleotideFAD
Flavin mononucleotideFMN
1 4 532 6 7 8 10 119 12 13 14 1615 17 18 19 2021
WC-1 +FADWC-1
WC-2 WC-2 +FAD
D LD L
D LD L
D L
D L
D L
D LD LD L
D L
D L
D L
L D
L
D
WC-1, bound to FAD, is the Neurospora circadian photoreceptor and does not require WC-2 for photoreception
FAD
FAD is required -compare 11 thru 16
WC-1 is the receptor -compare 20 vs 19
Both WC-1 and WC-2 are needed - lanes 1-10
How Does a Circadian Clock Work?
Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 29
How are light signals perceived and transduced to the clock?
•frq transcript is rapidly and highly induced in response to light.
•The frq promoter contains two cis-acting Light Response Element (LREs).
• The LREs are bound by a WC-1/WC-2 (WCC) containing complex.
• Light causes decreased mobility of the WCC/LRE.
proximalLRE
distalLRE
+1
frq promoter
? ?