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Molecular Mechanisms of Circadian Clocks

Molecular Mechanisms of Circadian Clocksallele linkage other clock properties arg-13 cel Clock Genes mitochondrially related cyb-3 cyt-4 olir phe-1 * * * frq VII R arrhythmic/ recessive

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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 locomoteractivity

Body temperature cycle

Circadian rhythm of melatonin secretion

Circadian rhythms of photosynthetic genes in plants

Circadian rhythm of asexual reproduction in fungiCircadian rhythm of asexual reproduction in fungi

24 hours ofgrowth

one circadian cyclePoint ofinoculation

side

top

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?

AB

CD

E OUTPUTINPUT gene expressionenzyme activityhormone release

LightTemperature

OSCILLATOR

Chemicals developmentbehavioractivity cyclesleep/wake cyclebody temperature

frqfrqfrqfrqfrqfrq temperature compensation,

period length dominance/recessivity affected

linkageallele other clock properties

temperature compensation,frqfrq

1

2346

7

89 arrhythmic/ recessive

temperature compensation

uncompensatedentrainmentnutritional compensation,

cycloheximide resetting

- isolated in the context of circadian rhythms

period length dominance/recessivity affected

linkageallele other clock properties

arg-13cel

Clock Genes

mitochondriallyrelated

cyb-3cyt-4olir

phe-1

*

*

*

temperature compensation,frq VII R arrhythmic/ recessiveuncompensated

entrainmentnutritional compensation,*

10

Mutations Affecting the Neurospora Clock

*

*

*

***

*

*

*

Other mutations affecting clock period length

21 - 19 recessiveI Rsemidominant16 hrsVII R20 - 40 temperature compensationIV Rsemidominant19.3 hrsVII R

cys-4 19IV Rsemidominant te24 hrsVII R mperature compensationcys-12 19I Rsemidominant19.3 hrsVII R

semidominant19.3 hrsVII R glp-3(ff-1) II R 19semidominant29 hrsVII R I L 19 recessive

VII R 29 hrs semidominantVII R

[mi-1] 18 - 19cya -5 IV R 19cyb-2 18

II L 20semidominantI R 20semidominant temperature compensationVI L 23.5 hrschr

VII R 18 - 20recessive temperature compensationprd-1 III C 25.8 hrsrecessiveprd-2 V R 25.5 hrsrecessive temperature compensationprd-3 I C 25.1 hrsdominantprd-4 I R 18 hrs temperature compensation

cla-1 I R/VII R 27 hrs semidominant temperature compensation

*Extent of period effect is dependent on the growth mediumGenetically frq2 = frq4 = frq6 and frq7 = frq8 **

Common theme among circadian oscillators

Negative Element(s)clock gene (s)

PositiveElement(s) (-)

(+)

Negative elements in circadian feedback loops:Synechococcus: kaiCNeurospora: FREQUENCYDrosophila: PERIOD and TIMELESS Mouse: mCRYs and mPERs

Positive elements in circadian feedback loopsSynechococcus: kaiANeurospora: WHITE COLLAR-1 and WC-2Drosophila: dCLOCK and CYCLE Mouse: CLOCK, NPAS2 and BMAL1

PAS domain containingtranscription 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

FRQ ORFsupstream ORFs

989aa

bp

basic serine rich/acidic acidicTG/SG repeat

coiled-coil1 kproline rich

frq 3 frq 7,8 frq 1 frq 91 bp deletion

frq 2,4,6ala895->thrgly459->asp gly482->serglu364->lys

(19.0 hr)(29.0 hr) (16.5 hr) (arrhythmic)(24.0 hr)

Both FRQ protein and frq RNA cycle

f r q + m R N A

2 0 3

1 1 8

F R Q

1 1 1 8 2 5 4 7D D 3 3 4 01 5 2 2 2 9 3 6 4 3 5 1 1 81 5 h o u r sC T 0 8 1 6 1 6 4 84 1 2 2 0 0 4 1 2 2 08

f r q + f r q 1 0

0

5

1 0

1 1 1 5 1 8 2 2 2 5 2 9 3 3 3 6 4 0 4 3 4 7 5 1H o u r s i n C o n s t a n t D a r k n e s s

5

1 0

0

The Neurospora (fungal) circadian feedback loops

FRQ

WC-1/WC-2

frq

+

+

WC-1

WC-2

-

+

Circadian interlocked feedback loops in fruit fly and mouse

mCrysmPers

+

+Bmal1CLOCK/NPAS2

-BML/CLK/NPAS2

mCRYsmPERs

Mammals

dPerTim

+

+dClock

Cycle

-CLK/CYC

PERTIM

Fruit fly

The positive feedback loops are important for the robustness of the clock

FRQ

Time Time

low levels of WCs high levels of WCs

How do environmental signals reset clocks?

Two most important signals: Light and Temperature

Circadian rhythm of locomoteractivity

Light resetting of the Neurospora clock

DD

LD12/12

Light resets the Neurospora clock by induction of frq mRNA

t i m e

f r q R N A

f r q R N A

hours in dark- light

frq

0

10

20

30

40

50

60fr

q

ribos

omal

RN

A

28 32 36 44 48 28 32 36 44 4840 40

+ light

- light + lighthours in the dark before a 2 minute light pulse

28 32 36 40 44 48 28 32 36 40 44 48

Light resets the Drosophila clock by degrading TIMELESS protein

Light resets the mammalian clock by inducing Per RNA expression

Dark

Light

Cell

NucleusFRQ

PP

kinase

turnover

?

frq

ccgs

CCREfrq

CCREccgs

Clock-controlledprocesses

FRQ

Cell

Nucleus

CYC:CLK

PER

PP

turnover?

per

ccgsCCREccgs

Clock-controlledprocesses

TIM/PER tim

E-boxTIM

E-box

DBT

FungiLIGHT

WC-2::WC-1

-+ Insect