The evidence for the impact of the transcriptional feedback loop on the clock is substantial. But as with many scientific problems, there are sometimes puzzling inconsistencies, experiments that don't squarely fit with the hypothesis. It doesn't mean that anything is wrong, but it means that we need to figure out how to accommodate the data. It's a puzzle that needs to be solved. Something that's perfect for scientists, right? So what are the exceptions to the rule that I'm referring to? Acetabularia is a giant algae. It consists of three structures. The rhizoid or the roots. The stem which can be up to ten centimeters long. And a cap, which is sort of like an umbrella that serves to spread chloroplasts around a large area so that a lot of energy can be harvested through photosynthesis. Like with higher plants that live on dry land, photosynthesis shows a profound circadian rhythm in acetabularia with all the machinery up regulated during the day time, even in constant light conditions. This entire acetabularia plant is made from a single cell, which means that it has a single nucleus. The nucleus sits down in the roots. When the roots are cut off, the circadian rhythm and photosynthesis continues on as though nothing had changed, but no nucleus means no transcription. So the puzzle is, how might this occur? Another cell without a nucleus that shows a circadian rhythm is the human red blood cell. In this case, an oscillation in the redox state of a small molecule called peroxiredoxin shows a circadian rhythm. This means that it's gaining electrons or losing them depending on what time of day it is. Many proteins are found in either an oxydized or a reduced state again impacting their function. In this case, it's not assumed that this molecular reaction is the engine of the clock but rather that it's reflecting organization of reducing and oxidizing processes in the cell according to time of day. Processes that continue on, even without a nucleus. There are many rhythms that persist in neurospora crassa without the FRQ protein. One of these is the same peroxiredoxin example. Its redox state oscillates when FRQ is present with the same period as other molecular oscillations in the cell. Without FRQ an oscillation takes slightly longer though it still occurs with a period closer to 28 hours. When the conidiation rhythms are measured in FRQ-less mutants conidiation in the spore formation that we saw in raised tubes. When these are measured without FRQ they look to be about the same period as a wild type strain. But the rhythm is very weak and hard to see through lots of non rhythmic conidiation. By pairing the FRQ mutation with certain mutations in metabolic pathways however, a whole collection of very long and robust oscillations in conidiation emerge. Sometimes these are very, very long. We don't even call them circadian, 50, 60, or 70 hours, and they're not temperature compensated. So although the clock is clearly very damaged, this suggests that there's still machinery that can put together self sustained oscillations. Continuing on with the neurospora example, when the non rhythmic FRQ mutants, the ones that don't make a working FRQ protein, are put into entraining conditions, specifically using temperature cycles, they entrain like a circadian clock, although they entrain their conidiation rhythm to the wrong time of day entirely. Possibly, the most inspiring example of a circadian rhythm without transcription is the famous clock in a tube experiment. I haven't said much yet about the prokaryotic circadian clock, the clock in bacteria. All of the examples that I've discussed so far have concerned eukaryotes, the animal-plant-fungi lineages. But in the first lecture I told you about how strategic some of the cyanobacteria were to compartmentalize nitrogen fixation and photosynthesis to opposite times of day, so that they don't need to make a physical compartment in this cell. That obviously takes some pretty exquisite temporal programming. Does their circadian clock work with a negative feedback loop also? Apparently not, or at least not in the same formulaic construction that we saw for the eukaryotic model systems. Yes, the cyanobacteria clock uses transcription as a key mechanism to organize the cell, but here the feedback loop mechanism is based on phosphorylation of a protein called KaiC. KaiC is highly phosphorylated during the subjective night and weakly phosphorylated during the subjective day. These rhythms in phosphorylation are remarkably robust and easy to follow by running the proteins in a gel like I showed you earlier for other clock proteins. The phosphorylation rhythm in KaiC came to be used as a marker for the circadian clock, going up and down each 24 hours. Using mutant screens, it was shown that this phosphorylation depends on two other proteins, KaiA which promotes KaiC phosphorylation, and KaiB which promotes its dephosphorylation. All three of these proteins are essential for the clock for determining free running period. For instance, but KaiC is is the protein that's regulating transcription according to its phosphorylation state. The clock in the tube experiment took purified KaiA, KaiB, and KaiC proteins, put them into a tube with ATP as a source of phosphate, for phosphorylation, and followed the state of KaiC for many days. Just to be clear, three proteins, ATP, and no cells. Incredibly, the oscillation and phosphorylation continues on with a circadian rhythm, even outside of a cell. It behaves a lot like an intact circadian system because it's temperature compensated, even in the tube. And if you take protein from mutant strains that have defective temperature compensation, then this is defective in the tube also. Some experiments were even done using temperature pulses as a zeitgeber allowing the system to synchronize in the tube. This experiment showed that the clock in this cyanobacteria, synechococcus, can be reconstituted a) without transcription, and b) with remarkably few molecular components. This is in stark contrast to the complexity that all of eukaryotic systems seem to have built into their circadian timing programs. How to reconcile all of this? We're still discussing this as a field, sometime with quite some difficulty. It's just a wonderful problem set. What a lot of great data. [SOUND].
