W12L2 The circadian clock in Drosophila Flashcards
What is a circadian rhythms
-Biologicla rhythms of a day that are controlled by an internal oscillator
Circadian rhythms in human
-at 9pm, melatonin secretion cause sleepiness
-stop of melatonin secretion in 07:30
-but there is genetic variation on the circadian clock
Evidence of Self sustaining circadian clock
-release animal in a 24h of free run of light dark cycle or continuous dark
- have active and inactive period as if it is in a normal day. But there is shift in time due to circadian clock being faster
How Circadian rhythms anticipate predictable daily environment
Young sunflowers reposition during the night to face the rising sun
Circadian rhythms of gene expression
-circadian rhythm of gene expression with promoter: luciferase
-5-90% of gene are under circadian regulation. Half the gene on average
Circadian clocks are entrained by the environment
-environmental input goes through an oscillator which lead to output
-Zeitgebers (time-keepers) such as light and temperature adjust the phase of the circadian oscillator
Expirement on Light as a zeigeber (timekeeper)
- a normal organism is put into a all dark environment with pulse of light given
-measure the oscillation
-If light is given in the subjective day time, noting happen
-if light is given at early night time, delay in ossicaltion. Late in the night, advance oscillation
-entrainment allows organism to adapt to changing day length
Circadian master clock
-In animals, ‘master’ clocks synchronise peripheral clocks
-photic zeitgeber influence master clock
-non photic zeitgeber influence peripheral and cellular osicillator
-synchronization occur
What is a circadian oscillator?
Transcription-translation feedback loops (TTFL)
How is the 24h cycle mainatin
-translational repression
-Codon bias
-Nuclear localization
-Phosphorylation
-Protein degration
-Transcriptional repression
-ncRNA
Period, the first circadian mutant
- 3 alleles in the period mutant: per0 active all time, pers short period mutant, perl long droid mutant
-3 mutant mapped to X chromosome
-complementation test show that :per0 is null mutant, pers and perl is missense mutatant
How does period gene works
-period forms a negative feedback loop
-a lag between the protein level and transcript abundant
-introducing the period gene (only coding region) can restore the oscillation can restore per0back to WT> period is self regulating
timeless, a second circadian mutant
-rhythmic transcription
- tim arrhythmic mutant does not have the oscillation transcription
Hyphothesis on tim and per
TIM and PER form a dimer to make a negative feedback loop to repress their own transcription
-TIM is needed for nuclear localization
-both tim and per does not have a DNA binding domain
-to additional arrhythmic mutants was discovered: clock and cycle, they are transcriptional activator of tim and per by binding to an E box
Post transcriptional regulation in the circadian clock
1/ phosphorylation/ dephosphorylation
2/ protein degration
3/ translational control
Phosphorylation regulation in the circadian clock
- phosphorylation / dephosphorylation: DBT (double time) is a kinase, phosphorilation of per allow it to interact with TIM
-TIM and Per complex need to be phosphorolyse to enter the nucleases
-SGG and CK2 phosphorylation TIM and PER (allow localization)
-DBT phosphorylation CLK> release CLK CYC from e box - Protein phosphatases (PP1 and PP2a) also act on TIM and PER
-PP1/PP2a counteract SSG2/CK to inhibit nuclear localization of TIM-PER-DBT
-acts as a speed bump to slow circadian clock
Light-dependent degradation of TIM by Ubiquitin Proteasome
-Crytochrome is a blue light receptor, activation lead to interaction with TIM, cause TIM to disassociate with PER
-Crytochrome after use is degraded by JETLAG
-Jetlag is a F box which target Crytochrome for degradation by ubiquitination
-a phosphorylation event allow JET to interact with TIM
-Target of TIM for degradation, after that, DBT phosphorylation PER, causing it to be regconise by another F-box slimB. degredation of pER by UPS
-CLK-CYC can now bind to e box, activating transcription
translational regulation of the circadian clock
-there is a 6-8 hours lag between per mRNA and protein
-due to degradation and control of translation, (transcription promoter TWF)
-mutant TWF have lower PER rhythms
-works via associating the 5’ cap with the PABP, stabilize the mRNA structure to promote translation
