10€ per copy, basically a steal

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Bonus! DnaA is also a transcription factor, so together with @baybioms.bsky.social we also investigated how changes in its regulation alter E. coli proteome! Surprisingly, impacting titration or switch alone seems to have no impact on the genes known to be regulated by DnaA...

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There is still a long road ahead in characterizing the role of initiator titration in controlling DNA replication. Still, it's exciting how cutting-edge microscopy techniques allow us to tackle decades-old biological problems!
Read more at: doi.org/10.1038/s414...

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More active DnaA in the cell? The chromosome titrates DnaA more, minimizing re-initiations. Less active DnaA? More DnaA is free, mitigating delays in replication. Even more interestingly, severely hampering titration leads to frequent re-initiation events!

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We then deleted known DnaA control loci and measured the DNA content of cells through flow cytometry, while we used microscopy 🔬 to measure their size and to determine the bound fraction of DnaA. We could then suggest a role of titration in stabilizing DNA replication in slow-growing E. coli.

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Next, using single-particle tracking 🔬, we observed the binding state of DnaA in live E. coli cells during steady-state growth at different growth rates. Consistent with previous modelling, we observed that the chromosome of E. coli controls DnaA bound fraction in a growth rate-dependent fashion!

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We started by checking the distribution of DnaA boxes on the chromosome of E. coli. Glad to say that @stephkoe.bsky.social‬ totally knocked it out of the park with this analysis! He showed that DnaA boxes tend to be enriched around oriC, a favorable feature for initiator titration.

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However, recent experimental and modelling efforts suggest that only a concerted action of switch and titration can generate stable cell cycles. Still, to date there is no direct evidence of DnaA being titrated on the chromosome of E. coli. We decided to try to tackle this gap.

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We now know that DnaA exists in two forms: DnaA-ATP, competent in unwinding oriC and DnaA-ADP, inactive in oriC unwinding. In this switch model, it is the accumulation of DnaA-ATP that determines replication initiation. As a result, the switch model almost replaced initiator titration.

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During early days of characterization of DNA replication control, it was hypothesized that the chromosome could act as a sponge 🧽 for DnaA, retarding DnaA binding to oriC by first titrating it on the high affinity boxes. This form of control is what is known as initiator titration model.

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DnaA binds DNA in specific 9 bp motifs called DnaA boxes. These boxes can have high or low affinity, depending on how well they match a specific consensus sequence. There are hundreds of high affinity boxes spread across the chromosome of E. coli. However, oriC mostly contains low-affinity motifs.

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In E. coli, DNA replication starts when DnaA binds to the origin of replication oriC and unwinds it, allowing for the rest of the DNA polymerase to assemble. This initiation step must be finely regulated to ensure that DNA replication happens timely and only once per cell cycle.

Ready for some sweet long(er) tracks finally!

We indeed already played around with maintaining cells at fixed density for live-cell imaging in a couple of studies now, but only recently moved to the Pioreactor and it was such a big improvement!

Feel like "Creep" also definitely deserves a spot! Such an unnerving one