Transcription factor search for a DNA promoter in a three-states model

To ensure fast gene activation, Transcription Factors (TF) use a mechanism known as facilitated diffusion to find their DNA promoter site. Here we analyze such a process where a TF alternates between 3D and 1D diffusion. In the latter (TF bound to the DNA), the TF further switches between a fast translocation state dominated by interaction with the DNA backbone, and a slow examination state where interaction with DNA base pairs is predominant. We derive a new formula for the mean search time, and show that it is faster and less sensitive to the binding energy fluctuations compared to the case of a single sliding state. We find that for an optimal search, the time spent bound to the DNA is larger compared to the 3D time in the nucleus, in agreement with recent experimental data. Our results further suggest that modifying switching via phosphorylation or methylation of the TF or the DNA can efficiently regulate transcription.Comment: 4 pages, 3 figure

Proteinaceous microspheres for targeted RNA delivery prepared by an ultrasonic emulsification method

In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the “ultrasonic emulsification method”. It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA–RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA–BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA–BSA complex are reported.Ulyana Shimanovich thanks Ministry of Science and Technology, Israel for the "Woman in Science" scholarship (3-8219)

Acting on impulse: dissecting the dynamics of the NFAT transcriptional response

Examining NFAT transcription factor dynamics in single cells during signal propagation from cell membrane to the nucleus reveals unique modes of operation for protein isoforms

Quantitative analysis of chromatin compaction in living cells using FLIM-FRET

FRET analysis of cell lines expressing fluorescently tagged histones on separate nucleosomes demonstrates that variations in chromosome compaction occur during mitosis

Cell-to-cell variability of alternative RNA splicing

The role of mRNA processing in gene expression variability is poorly characterized. This study investigates the extent of cell-to-cell variability of alternative RNA splicing in mammalian cells using single-molecule imaging of CAPRIN1 and MKNK2 splice isoforms

Assembling an intermediate filament network by dynamic cotranslation

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these “translation factories.” This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation

Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli

Robustness of circadian clocks to daylight fluctuations: hints from the picoeucaryote Ostreococcus tauri

The development of systemic approaches in biology has put emphasis on identifying genetic modules whose behavior can be modeled accurately so as to gain insight into their structure and function. However most gene circuits in a cell are under control of external signals and thus quantitative agreement between experimental data and a mathematical model is difficult. Circadian biology has been one notable exception: quantitative models of the internal clock that orchestrates biological processes over the 24-hour diurnal cycle have been constructed for a few organisms, from cyanobacteria to plants and mammals. In most cases, a complex architecture with interlocked feedback loops has been evidenced. Here we present first modeling results for the circadian clock of the green unicellular alga Ostreococcus tauri. Two plant-like clock genes have been shown to play a central role in Ostreococcus clock. We find that their expression time profiles can be accurately reproduced by a minimal model of a two-gene transcriptional feedback loop. Remarkably, best adjustment of data recorded under light/dark alternation is obtained when assuming that the oscillator is not coupled to the diurnal cycle. This suggests that coupling to light is confined to specific time intervals and has no dynamical effect when the oscillator is entrained by the diurnal cycle. This intringuing property may reflect a strategy to minimize the impact of fluctuations in daylight intensity on the core circadian oscillator, a type of perturbation that has been rarely considered when assessing the robustness of circadian clocks