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

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

Problem Unanswered questions regarding how the circadian clock mechanism in Cyanobacteria works In particular, the clock resetting mechanism in Cyanobacteria is not well known. Hypothesis It 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., 2005

Results If Pex does not interact with the Kai proteins, we should expect no change in the circadian rhythm of KaiC phosphorylation in vitro. % of Phosphorylated KaiC Hours Pex added here

Results If Pex interacts with the Kai proteins, we should expect a phase delay in the circadian rhythm of KaiC phosphorylation. Hours % of Phosphorylated KaiC Pex added here

Future steps: For either negative or positive results: Measure transcription using Northern Blots Further 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 Nov 10 M. Nakajima et al., Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science Apr 15; 308(5720): T. Nishiwaki et al., Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC Proc Natl Acad Sci U S A Sep 21; 101(38): 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 Aug 8; 103(32): 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 Aug ; 21(4): J Tomita et al., No transcription-translation feedback in circadian rhythm of KaiC phosphorylation. Science Jan 14; 307(5707): 251-4