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CYANOBACTERIA CLOCK MCB 186 CIRCADIAN BIOLOGY December 13, 2006 Hetmann Hsieh Proposal to Determine the Posttranslational Effect of Circadian Clock–Resetting Gene pex and the Pex Protein

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CYANOBACTERIA CLOCK. Proposal to Determine the Posttranslational Effect of Circadian Clock–Resetting Gene pex and the Pex Protein. MCB 186 CIRCADIAN BIOLOGY. December 13, 2006 Hetmann Hsieh. Overview of the Circadian System in Cyanobacteria. - PowerPoint PPT Presentation

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Page 1: CYANOBACTERIA CLOCK

CYANOBACTERIA CLOCK

MCB 186CIRCADIAN BIOLOGY

December 13, 2006Hetmann Hsieh

Proposal to Determine the Posttranslational Effect of Circadian Clock–Resetting Gene pex and the Pex ProteinProposal to Determine the Posttranslational Effect of Circadian Clock–Resetting Gene pex and the Pex Protein

Page 2: CYANOBACTERIA CLOCK

Overview of the Circadian System in Cyanobacteria

KaiA, KaiB, and KaiC interact to produce oscillation in the phosphorylation state of KaiC

Proposed model of Kai system

Tomita et al, 2005

Page 3: CYANOBACTERIA CLOCK

Overview of the Circadian System in Cyanobacteria

Reconstitution of KaiA, KaiB, and KaiC proteins in vitro

(Nakajima et al., 2005)

Page 4: CYANOBACTERIA CLOCK

Background of Circadian Clock-Related pex Gene

Period extender (pex) mutants have a short-period phenotype Free running period of

pex mutant is about 1 hour shorter than the wild-type cell

Pex is classified as a resetting-related gene

Takai et al, 2006

Page 5: CYANOBACTERIA CLOCK

Background of Circadian Clock-Related Pex protein

Structure of Pex shows that it has a DNA-binding motif Winged-helix motif

In vivo assays

Arita et al, 2006Takai et al, 2006

Page 6: CYANOBACTERIA CLOCK

ProblemUnanswered questions regarding how the circadian

clock mechanism in Cyanobacteria worksIn particular, the clock resetting mechanism in

Cyanobacteria is not well known.

HypothesisIt is possible that Pex regulates the Cyanobacteria

oscillator proteins posttranscriptionally. Any interaction of the Pex protein with the Kai proteins can be determined by combining the proteins in vitro.

Page 7: CYANOBACTERIA CLOCK

Proposed Experiment:Repeat Nakajima et al. in vitro experiment as

a negative control.Purify Pex protein and incubate Pex with

KaiA, KaiB, and KaiC.Incubate twice the initial amount of Pex

protein with the Kai system in vitro.Incubate four times the initial amount of Pex

protein with the Kai system in vitro.

Page 8: CYANOBACTERIA CLOCK

Methods Produce KaiA, KaiB, KaiC, and Pex proteins in E. Coli

The Kai genes can be found in the following BioBricks: BBa_J36831 contains the coding region of KaiA BBa_J36832 contains the coding region of KaiB BBa_J36834 contains the coding region of KaiC

Perform extraction of Pex coding region Purify these proteins using method by Nishiwaki, 2004 Incubate KaiA (0.05 μg/μl), KaiB (0.05 μg/μl), and KaiC (0.02 μg/μl), in reaction

buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM ATP) at 30°C

Incubate KaiA (0.05 μg/μl), KaiB (0.05 μg/μl), KaiC (0.02 μg/μl), and Pex (0.02 μg/μl) in reaction buffer at 30°C

Incubate KaiA (0.05 μg/μl), KaiB (0.05 μg/μl), KaiC (0.02 μg/μl), and Pex (0.04 μg/μl) in reaction buffer at 30°C

Incubate KaiA (0.05 μg/μl), KaiB (0.05 μg/μl), KaiC (0.02 μg/μl), and Pex (0.08 μg/μl) in reaction buffer at 30°C

Collect aliquots (3-μl) of the reaction mixture every hour Stop the reaction by adding SDS sample buffer: (62.5 μM Tris-HCl, pH 6.8, 2%

SDS, 10% glycerol). Western blot samples using anti-KaiC antibodies Image Western blot

In vitro protocol courtesy of Nakajima et al., 2005In vitro protocol courtesy of Nakajima et al., 2005

Page 9: CYANOBACTERIA CLOCK

ResultsIf Pex does not interact with the Kai proteins, we should expect no change in the circadian rhythm of KaiC phosphorylation in vitro.

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late

d K

aiC

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Pex added herePex added here

Page 10: CYANOBACTERIA CLOCK

ResultsIf Pex interacts with the Kai proteins, we should expect a phase delay in the circadian rhythm of KaiC phosphorylation.

Hours

% o

f P

hosp

hory

late

d K

aiC

Pex added herePex added here

Page 11: CYANOBACTERIA CLOCK

Future steps:For either negative or positive results:

Measure transcription using Northern BlotsFurther structural analysis

For positive results:Pex could be tested in E. Coli in

synchronization experiments

Page 12: CYANOBACTERIA CLOCK

References K. Arita et al., Structural and biochemical characterization of a

cyanobacterium circadian clock modifier protein. J Biol Chem. 2006 Nov 10

M. Nakajima et al., Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science. 2005 Apr 15; 308(5720): 414-5

T. Nishiwaki et al., Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942. Proc Natl Acad Sci U S A. 2004 Sep 21; 101(38): 13927-32

N Takai et al., A KaiC-associating SasA-RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria. Proc Natl Acad Sci U S A. 2006 Aug 8; 103(32): 12109-14

N Takai et al., Expression of the circadian clock-related gene pex in cyanobacteria increases in darkness and is required to delay the clock. J Biol Rhythms. 2006 Aug ; 21(4): 235-44

J Tomita et al., No transcription-translation feedback in circadian rhythm of KaiC phosphorylation. Science. 2005 Jan 14; 307(5707): 251-4