Molecular Mechanisms
of Circadian Clocks
Circadian Rhythms
• Endogenous rhythm which persists under
constant conditions
• Period of approximately 24 hours
• Entrainable (reset) by environmental cues
such as light and temperature
• Temperature compensated - period stays
about the same at different temperatures
Circadian rhythm of locomoter
activity
Body temperature cycle
Circadian rhythm of melatonin
secretion
Circadian rhythms of photosynthetic
genes in plants
Circadian rhythm of asexual
reproduction in fungi
side
Point of
inoculation
one circadian cycle
top
24 hours of
growth
Neurospora
Suprachiasmatic Nucleus (SCN), the
Master Pacemaker in Mammals
Science 2001 Feb 9;291(5506):1040-1043
An hPer2 phosphorylation site mutation
in familial advanced sleep phase
syndrome (FASP).
Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM,
Ptacek LJ, Fu YH.
Department of Human Genetics, University of Utah
How does a clock work?
Light
Temperature
Chemicals
OSCILLATOR
A
INPUT
OUTPUT
E
B
D
C
gene expression
enzyme activity
hormone release
development
behavior
activity cycle
sleep/wake cycle
body temperature
Mutations Affecting the Neurospora Clock
Clock Genes
- isolated in the context of circadian rhythms
allele linkage
1
frq
*
frq2
frq3
*4
frq
*
frq6
*
frq7
*
frq8
frq9
frq 10
chr
prd-1
prd-2
prd-3
prd-4
cla-1
*
period length
dominance/
recessivity
other clock properties
affected
semidominant
semidominant
semidominant temperature compensation
semidominant
semidominant
semidominant temperature compensation,
cycloheximide resetting
VII R
29 hrs
semidominant temperature compensation
temperature compensation,
VII R
arrhythmic/ recessive
**
uncompensated
nutritional compensation,
entrainment
temperature compensation,
VII R
arrhythmic/ recessive
**
uncompensated
nutritional compensation,
entrainment
semidominant temperature compensation
VI L
23.5 hrs
recessive
temperature compensation
III C
25.8 hrs
recessive
VR
25.5 hrs
recessive
temperature compensation
IC
25.1 hrs
dominant
IR
18 hrs
temperature compensation
I R/VII R 27 hrs
semidominant temperature compensation
VII R
VII R
VII R
VII R
VII R
VII R
16 hrs
19.3 hrs
24 hrs
19.3 hrs
19.3 hrs
29 hrs
Genetically frq2 = frq4 = frq6 and frq7 = frq8
Other mutations affecting clock period length
allele
linkage period length
**
arg-13
**
cel
cys-4
cys-12
IR
IV R
IV R
IR
21 - 19
20 - 40
19
19
glp-3(ff-1) II R
19
phe-1
IL
mitochondrially
related
19
[mi-1]
cya -5
cyb-2
IV R
18 - 19
19
18
cyb-3
cyt-4
olir
II L
IR
VII R
20
20
18 - 20
dominance/
recessivity
other clock properties
affected
recessive
temperature compensation
recessive
semidominant
** Extent of period effect is dependent on the growth medium
Common theme among circadian
oscillators
Positive
Element(s)
(-)
(+)
Negative Element(s)
clock gene (s)
Negative elements in circadian feedback loops:
Synechococcus:
kaiC
Neurospora:
FREQUENCY
Drosophila:
PERIOD and TIMELESS
Mouse:
mCRYs and mPERs
Positive elements in circadian feedback loops
Synechococcus:
kaiA
Neurospora:
WHITE COLLAR-1 and WC-2
Drosophila:
dCLOCK and CYCLE
Mouse:
CLOCK, NPAS2 and BMAL1
PAS domain containing
transcription factors
Definitions of a central component
of a circadian clock
• Mutations result in long, short period, or
arrhythmic phenotypes
• Deletion of the locus abolishes the rhythms
• Both mRNA and protein cycle
• Constitutive expression eliminates the clock
• Feeds back negatively on its own expression
• Change in levels resets the clock
Transcripts and Domains in frq and FRQ
upstream ORFs
FRQ ORFs
1 kbp
989aa
coiled-coil
TG/SG repeat
basic
frq7,8
frq3
gly459->asp
glu364->lys
(29.0 hr)
(24.0 hr)
proline rich
serine rich/acidic
frq1
gly482->ser
(16.5 hr)
acidic
frq 9
1 bp deletion
(arrhythmic)
frq 2,4,6
ala895->thr
(19.0 hr)
Both FRQ protein and frq RNA cycle
frq +
C T
D D
0
4
8 1 2 1 6 2 0
1 1 1 5 1 8 2 2 2 5 2 9
0
4
8
3 3 3 6 4 0
frq 1 0
1 2 1 6 2 0
4 3 4 7 5 1
4
8
1 5 1 8
h o u rs
2 0 3
F R Q
1 1 8
frq + m R N A
1 0
1 0
5
5
0
1 1
1 5 1 8 2 2 2 5 2 9 3 3 3 6 4 0 4 3
H o u rs in C o n s ta n t D a rk n e s s
4 7
5 1
0
The Neurospora (fungal) circadian feedback
loops
WC-1
WC-2
WC-1/WC-2
+
frq
+
+
FRQ
Circadian interlocked feedback loops in
fruit fly and mouse
Bmal1
dClock
+
Cycle
CLK/CYC
+
-
dPer
Tim
Fruit fly
BML/CLK/NPAS2
PER
TIM
+
CLOCK/
NPAS2
+
-
mCrys
mPers
Mammals
mCRYs
mPERs
FRQ
The positive feedback loops are important
for the robustness of the clock
Time
low levels of WCs
Time
high levels of WCs
How do environmental
signals reset clocks?
Two most important signals: Light and Temperature
Circadian rhythm of locomoter
activity
Light resetting of the Neurospora clock
DD
LD12/12
Light resets the Neurospora clock
by induction of frq mRNA
+ light
- light
hours in dark
28
32
36
40
44
48
28
32
36
40
44
48
frq
50
40
30
20
48
44
40
36
32
28
48
44
40
36
0
32
10
28
ribosomal RNA
frq
60
- light
+ light
hours in the dark before
a 2 minute light pulse
frq
R N A
frq
R N A
tim e
Light resets the Drosophila clock by
degrading TIMELESS protein
Light resets the mammalian clock by
inducing Per RNA expression
Dark
Light
LIGHT
Fungi
+
WC-2::WC-1
P P
turnover
FRQ
kinase
FRQ
frq
CCREfrq
Insect
-
CYC:CLK
P P
TIM/PER
turnover
?
ccgs
CCREccgs
TIM
DBT
PER
Nucleus
Clock-controlled
processes
?
tim
E-box
per
E-box
ccgs
CCREccgs
Nucleus
Cell
Clock-controlled
processes
Cell