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.

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.

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.

A recently reported ''spinach'' system labels RNAs with an aptamer sequence that specifically binds a small molecule and induces its fluorescence for imaging and was applied to image highly abundant tRNA and rRNA. Whether this tag can be used to image relatively low abundant mRNAs without affecting their biological functions remains to be verified. In order to enhance the brightness of spinach system and apply it for imaging of RNAs of low abundance, here we report a spinach array with tandem aptamer sequences to amplify its signal, and apply it to mRNA live cell imaging. We find that the spinach array increases the fluorescence intensity by more than 10-fold, and does not affect mRNA translation and degradation.

17Oscillating gene expression is crucial for correct timing and progression 18 through cell cycle. In Saccharomyces cerevisiae, G1 cyclins Cln1-3 are 19 essential drivers of the cell cycle and have an important role for 20 temporal fine-tuning. We measured time-resolved transcriptome-wide 21 gene expression for wild type and cyclin single and double knockouts 22 over cell cycle with and without osmotic stress. Clustering of expression 23 profiles, peak-time detection of oscillating genes, integration with 24 transcription factor network dynamics, and assignment to cell cycle 25 phases allowed us to quantify the effect of genetic or stress 26 perturbations on the duration of cell cycle phases. Cln1 and Cln2 showed 27 functional differences, especially affecting later phases. Deletion of Cln3 28 led to a delay of START followed by normal progression through later 29 phases. Our data and network analysis suggest mutual effects of cyclins 30 with the transcriptional regulators SBF and MBF. 33Eukaryotic cell cycle is a highly ordered process, which can be divided into four distinct phases 34 during which a specific set of events take place: Cell growth (G1 and G2 phase), duplication and 35 segregation of DNA (S phase) and the division of the nucleus (M phase), finally leading to 36 cytokinesis. To enable and control the progression through the cell cycle, a subset of genes is 37 transcribed in an oscillating pattern. Amongst those, cyclins are key regulatory proteins, which 38 trigger all fundamental events of the cell cycle (Haase & Reed, 1999; Lu & Cross, 2010). Cyclins 39 activate cyclin dependent kinases (CDKs), leading to phosphorylation of specific target proteins, 40 which among other things, initialize the expression of the next wave of oscillating genes (for 41 simplification we will refer to the cyclin-CDK complexes just by the name of their cyclins). The 42 cyclins are functionally conserved across many species including mammals (Harashima et al, 43 2013), which makes understanding their functions even more relevant. 44During G1 phase, the initial part of the cell cycle, three cyclins in Saccharomyces cerevisiae are 45 necessary to successfully start the first regulatory events of the cell cycle: Cln1, Cln2 and Cln3. 46These G1 cyclins have been extensively studied and were found to fulfill specific functions but 47 also to be able to partly compensate for each other in knockout studies. However, loss of all 48 three cyclins at once is lethal for the yeast cells (Richardson et al, 1989), highlighting their 49 importance for controlling cell cycle progression. 50Cln3 is the first cyclin expressed during G1 phase, which, by inactivating the transcriptional 51 repressor Whi5, is responsible for cell size control and the initial expression of the G1/S regulon. 52Two hypotheses have been proposed for the mechanism of the Cln3-Whi5 interaction: Both the 53 retention of Cln3 in the endoplasmic reticulum with release in late G1 phase (Vergés et al, 2007; 54 Wang et al, 2004) and the size-dependent dilution of Whi5 ...