Signaling kinetics of cyanobacterial phytochrome Cph1, a light regulated histidine kinase.

Cyanobacterial phytochrome 1 (Cph1) is a red/far-red light regulated histidine kinase, which together with its response regulator (Rcp1) forms a two-component light signaling system in Synechocystis 6803. In the present study we followed the in vitro autophosphorylation of Cph1 and the subsequent phosphotransfer to Rcp1 in different ionic milieus and following different light treatments. Both processes were red/far-red reversible with activity manifested in the Pr ground state (in darkness or after far-red irradiation) and with strongest activities being exhibited in the presence of Mn(2+). In vivo and in vitro assembled holoproteins in the Pr state displayed at least 4-fold higher efficiencies (k(cat)/K(m)) for autophosphorylation and phosphotransfer than the apoprotein or the holoprotein at photoequilibrium in red light. The reduced activities observed following red light treatments were consistent with the Pfr state being enzymatically inactive. Thus, both the rate of kinase autophosphorylation and the rate of phosphotransfer regulate the phosphorylation state of the response regulator, consistent with the rotary switch model regulating accessibility of the histidine target.

A transcriptome-wide analysis deciphers distinct roles of G1 cyclins in temporal organization of the yeast cell cycle.

Oscillating gene expression is crucial for correct timing and progression through cell cycle. In Saccharomyces cerevisiae, G1 cyclins Cln1-3 are essential drivers of the cell cycle and have an important role for temporal fine-tuning. We measured time-resolved transcriptome-wide gene expression for wild type and cyclin single and double knockouts over cell cycle with and without osmotic stress. Clustering of expression profiles, peak time detection of oscillating genes, integration with transcription factor network dynamics, and assignment to cell cycle phases allowed us to quantify the effect of genetic or stress perturbations on the duration of cell cycle phases. Cln1 and Cln2 showed functional differences, especially affecting later phases. Deletion of Cln3 led to a delay of START followed by normal progression through later phases. Our data and network analysis suggest mutual effects of cyclins with the transcriptional regulators SBF and MBF.

Transcriptional timing and noise of yeast cell cycle regulators-a single cell and single molecule approach.

Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response necessitates expression reprogramming and cell cycle arrest. While previous studies are mostly based on bulk experiments influenced by synchronization effects or lack temporal distribution, time-resolved methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. Using smFISH, microscopy and morphological markers, we monitored mRNA abundances over cell cycle phases and calculated transcriptional noise for SIC1, CLN2, and CLB5, the main G1/S transition regulators in budding yeast. We employed mathematical modeling for in silico synchronization and for derivation of time-courses from single cell data. This approach disclosed detailed quantitative insights into transcriptional regulation with and without stress, not available from bulk experiments before. First, besides the main peak in G1 we found an upshift of CLN2 and CLB5 expression in late mitosis. Second, all three genes showed basal expression throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. Finally, exposing cells to osmotic stress revealed different periods of transcriptional inhibition for CLN2 and CLB5 and the impact of stress on cell cycle phase duration. Combining experimental and computational approaches allowed us to precisely assess cell cycle progression timing, as well as gene expression dynamics.