Scientists assemble a biological clock in a test tube to study how it works

A group of UC scientists recreated the circadian clock in cyanobacteria using a test tube. This allowed them to examine the molecular interactions between clock proteins in real-time and to understand how they control gene expression. Andy LiWang
Our cells have biological clocks, also known as circadian clocks. They drive daily cycles in almost every area of our physiology. The biological rhythms of our lives are kept in sync by the cyclical interactions between clock proteins. This is true not only for humans, but also for complex animals, such as mammals, and even single-celled organisms like cyanobacteria.

Scientists have now recreated the circadian clock in cyanobacteria using a test tube. This allows them to observe rhythmic interactions between clock proteins in real-time and to understand how they control gene expression. The study was published in Science on October 8th by scientists from three labs at UC Santa Cruz and UC Merced.

"Reconstructing a complex biological process such as the circadian clock from scratch has really helped us understand how clock proteins work together, and will enable a deeper understanding of circadian rhythms," Carrie Partch (UC Santa Cruz professor of chemistry, and a co-author of the study).

Partch pointed out that circadian clocks' molecular features are very similar between cyanobacteria and humans. A functioning clock can be studied in a test tube ("in vitro") rather than in living cells ("in-vivo"), which provides an excellent platform for studying the clock's mechanisms and responding to changes. To confirm their in vitro findings, the team performed experiments in living cells.

These results are so unexpected because it is not common for results to be inconsistent in vitro with those observed in vivo. Andy LiWang (UC Merced professor of chemistry, biochemistry, and corresponding author) said that the interior of live cells is complex in stark contrast with the simpler conditions found in vitro.

This new research builds upon previous Japanese researchers' 2005 work, which reconstructed the cyanobacterial circadian clock oscillator, the basic 24-hour timekeeping mechanism of the clock. The oscillator is composed of three proteins that are closely related: KaiA and KaiB. Signals from the oscillator are sent to living cells by other proteins. They control genes expression in a circadian cycle.

In vitro clocks now include, in addition the oscillator proteins and two kinase protein (SasA, CikA), whose activities can be modified by interfacing with the oscillator. Also included is a DNA binding protein (RpaA), and its target DNA.

LiWang said that SasA and CikA activate and deactivate RpaA in a way that rhythmically binds to and unbinds DNA. "In Cyanobacteria this rhythmic binding/unbinding at more than 100 sites in their genome activates or deactivates many genes that are important for health and survival."

Researchers used fluorescent labeling techniques to track interactions among all clock components, as the system oscillates in a circadian rhythm over many days or even weeks. The team was able to identify how CikA and SasA increase the stability of the oscillator. This allowed them to keep it ticking even under conditions where the KaiABC proteins would cease oscillating.

Researchers also used in vitro systems to study the genetic causes of clock disruption in an arrhythmic strain cyanobacteria. One mutation in RpaA's gene was identified that reduced the protein's DNA binding efficiency.

"A single amino acid alteration in the transcription factor causes the cell to lose the rhythm gene expression even though its clock remains intact," stated Susan Golden, coauthor and director of the Center for Circadian Biology, UC San Diego. Partch, LiWang, are also members.

She said that the real beauty of this project was how the three UC campuses came together and pooled their approaches to answering how a cell tells time. The active collaboration went beyond the principal investigators. Students and postdocs from different disciplines shared their genetics, structural biology and biophysical data and explained to each other the importance of their findings. Cross-discipline communication was just as important as the outstanding skills of the researchers for the success of this project.

Continue reading What makes circadian clocks tick

Information: Archana Chavan and colleagues, Reconstitution a intact clock reveals mechanisms for circadian timekeeping. Science (2021). Journal information: Science Archana Chavan et al, Reconstitution of an intact clock reveals mechanisms of circadian timekeeping,(2021). DOI: 10.1126/science.abd4453