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
Dec 19, 2015
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
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
Overview of the Circadian System in Cyanobacteria
Reconstitution of KaiA, KaiB, and KaiC proteins in vitro
(Nakajima et al., 2005)
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
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
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.
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.
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
ResultsIf Pex does not interact with the Kai proteins, we should expect no change in the circadian rhythm of KaiC phosphorylation in vitro.
% o
f P
hosp
hory
late
d K
aiC
Hours
Pex added herePex added here
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
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
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