Reconstituting the cyanobacterial circadian clock in vitro

Author

Pyong Hwa Kim, New Jersey Institute of Technology

Document Type

Dissertation

Date of Award

5-31-2021

Degree Name

Doctor of Philosophy in Chemistry - (Ph.D.)

Department

Chemistry and Environmental Science

First Advisor

Yong Ick Kim

Second Advisor

Edgardo Tabion Farinas

Third Advisor

Tamara M. Gund

Fourth Advisor

Yuanwei Zhang

Fifth Advisor

Casey Diekman

Abstract

Cyanobacteria are photosynthetic organisms that are known to be responsible for oxygenating Earth’s early atmosphere. Having evolved to ensure optimal survival in the periodic light/dark cycle on this planet, their genetic codes are packed with various tools, including a sophisticated biological timekeeping system. Among the cyanobacteria is Synechococcus elongatus PCC 7942, the simplest clock-harboring organism with a powerful genetic tool that enabled the identification of its intricate timekeeping mechanism. The three central oscillator proteins—KaiA, KaiB, and KaiC—drive the 24 h cyclic gene expression rhythm of cyanobacteria, and the "ticking" of the oscillator can be reconstituted inside a test tube just by mixing the three recombinant proteins with ATP and Mg^2+.

Along with its biochemical resilience, the post-translational rhythm of the oscillation can be reset through sensing oxidized quinone, a metabolite that becomes abundant at the onset of darkness. In addition, the output components pick up the information from the central oscillator, tuning the physiological and behavioral patterns and enabling the organism to better cope with the cyclic environmental conditions. In this research, how the cyanobacterial circadian clock functions as a molecular chronometer that readies the host for predictable changes in its surroundings is highlighted and discussed.

Since the bottleneck of performing any in vitro experiments is the laborious task of purifying proteins with enough purity, the most efficient method to extract KaiC is introduced, so that other impurities cannot hinder the highly sensitive post-translational activities. Next, CikA, an input component that synchronizes the oscillator to the environmental cues by using a metabolite called “quinone” as a proxy for darkness, is introduced. Finally, KaiC’s C-terminal linear chain undergoes conformational changes that determine the rising or falling phase of the clock. By creating site mutations that constitutively maintain the loop’s exposed conformation, a potentially additional role of another oscillator component, KaiB, is elucidated. Thus, by using the in vitro techniques that are compatible with this bacterial clock, the functional details that lie behind the biochemical workings of the circadian clock are disclosed.

Recommended Citation

Kim, Pyong Hwa, "Reconstituting the cyanobacterial circadian clock in vitro" (2021). Dissertations. 1516.
https://digitalcommons.njit.edu/dissertations/1516