Daily cycles in virtually every aspect of our physiology are driven by biological (or circadian) clocks in our cells. A team of interdisciplinary scientists has now reconstituted the circadian clock of cyanobacteria in a test tube, enabling them to study rhythmic interactions of clock proteins in real time, to help understand how these interactions enable the clock to exert control over gene expression. “Reconstituting a complicated biological process like the circadian clock from the ground up has really helped us learn how the clock proteins work together and will enable a much deeper understanding of circadian rhythms,” said Carrie Partch, PhD, professor of chemistry and biochemistry at University of California (UC) Santa Cruz and a corresponding author of the study, which is published in Science. The research team, from three labs at UC Santa Cruz, UC Merced, and UC San Diego describe their findings in a paper titled, “Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.

The cyclical interactions of clock proteins keep the biological rhythms of life in tune with the daily cycle of night and day, and this happens not only in humans and other complex animals, but even in simple, single-celled organisms such as cyanobacteria. “Circadian clocks are intracellular systems that provide organisms with an internal representation of local time and have profound consequences to health,” the authors wrote. And, as Partch further noted, the molecular details of circadian clocks are remarkably similar from cyanobacteria to humans.

The new study builds on previous work by Japanese researchers, who in 2005 reconstituted the cyanobacterial circadian oscillator, the basic 24-hour timekeeping loop of the clock. The oscillator consists of three related proteins: KaiA, KaiB, and KaiC. In living cells, signals from the oscillator are transmitted through other proteins to control the expression of genes in a circadian cycle. “The core circadian clock genes of cyanobacteria, kaiA, kaiB, and kaiC, are essential for rhythmic gene expression, and their proteins generate an autonomous ~24-hour rhythm of KaiC phosphorylation in vivo, which can be recapitulated in vitro,” the authors noted.

The new in vitro clock (IVC) includes, in addition to the oscillator proteins, two kinase proteins (SasA and CikA), whose activities are modified by interacting with the oscillator, as well as a DNA-binding protein (RpaA) and its DNA target. “SasA and CikA respectively activate and deactivate RpaA such that it rhythmically binds and unbinds DNA,” explained corresponding author Andy LiWang, PhD, professor of chemistry and biochemistry at UC Merced. “In cyanobacteria, this rhythmic binding and unbinding at over 100 different sites in their genome activates and deactivates the expression of numerous genes important to health and survival.”

Using fluorescent labeling techniques, the researchers were able to track the interactions between all of these clock components, as the whole system oscillates with a circadian rhythm for many days and even weeks. “The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention,” the investigators reported. They were then able to determine how SasA and CikA enhance the robustness of the oscillator, keeping it ticking under conditions in which the KaiABC proteins by themselves would stop oscillating.

A team of UC scientists reconstituted the circadian clock of cyanobacteria in a test tube, enabling them to study the molecular interactions of the clock proteins in real time and understand how these interactions enable the clock to exert control over gene expression. [Andy LiWang]

The researchers also used the in vitro system to explore the genetic origins of clock disruption in an arrhythmic strain of cyanobacteria. They identified a single mutation in the gene for RpaA that reduces the protein’s DNA-binding efficiency. “A single amino acid change in the transcription factor makes the cell lose the rhythm of gene expression, even though its clock is intact,” said coauthor Susan Golden, director of the Center for Circadian Biology at UC San Diego, of which Partch and LiWang are also members.

Having a functioning clock that can be studied in the test tube instead of in living cells provides a powerful platform for exploring the clock’s mechanisms and how it responds to changes. The team conducted experiments in living cells to confirm that their in vitro results are consistent with the way the clock operates in live cyanobacteria. “These results were so surprising because it is common to have results in vitro that are somewhat inconsistent with what is observed in vivo, said LiWang. “The interior of live cells is highly complex, in stark contrast to the much simpler conditions in vitro.”

The authors concluded,“The expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations … This newly developed IVC system offers an unparalleled opportunity to explore how the clock maintains consistency in vivo despite rhythmic changes in the concentration of clock components that result from the associated transcription translation feedback and protein turnover and provides an experimental platform for integrating the oscillator with the upstream and downstream components with which it interacts.”

“The real beauty of this project is how the team drawn from three UC campuses came together to pool approaches toward answering how a cell can tell time,” Golden added. “The active collaboration extended well beyond the principal investigators, with the students and postdocs who were trained in different disciplines conferring among themselves to share genetics, structural biology, and biophysical data, explaining to one another the significance of their findings. The cross-discipline communication was as important to the success of the project as the impressive skills of the researchers.”