In Escherichia coli , tetracycline prevents translation. When subject to tetracycline, E. coli express TetA to pump it out by a mechanism that is sensitive, while fairly independent of cellular metabolism. We constructed a target gene, P tetA -mRFP1-96BS, with a 96 MS2-GFP binding site array in a single-copy BAC vector, whose expression is controlled by the tetA promoter. We measured the in vivo kinetics of production of individual RNA molecules of the target gene as a function of inducer concentration and temperature. From the distributions of intervals between transcription events, we find that RNA production by P tetA is a sub-Poissonian process. Next, we infer the number and duration of the prominent sequential steps in transcription initiation by maximum likelihood estimation. Under full induction and at optimal temperature, we observe three major steps. We find that the kinetics of RNA production under the control of P tetA , including number and duration of the steps, varies with induction strength and temperature. The results are supported by a set of logical pairwise Kolmogorov-Smirnov tests. We conclude that the expression of TetA is controlled by a sequential mechanism that is robust, whereas sensitive to external signals.

BackgroundIn Escherichia coli the mean and cell-to-cell diversity in RNA numbers of different genes vary widely. This is likely due to different kinetics of transcription initiation, a complex process with multiple rate-limiting steps that affect RNA production.ResultsWe measured the in vivo kinetics of production of individual RNA molecules under the control of the lar promoter in E. coli. From the analysis of the distributions of intervals between transcription events in the regimes of weak and medium induction, we find that the process of transcription initiation of this promoter involves a sequential mechanism with two main rate-limiting steps, each lasting hundreds of seconds. Both steps become faster with increasing induction by IPTG and Arabinose.ConclusionsThe two rate-limiting steps in initiation are found to be important regulators of the dynamics of RNA production under the control of the lar promoter in the regimes of weak and medium induction. Variability in the intervals between consecutive RNA productions is much lower than if there was only one rate-limiting step with a duration following an exponential distribution. The methodology proposed here to analyze the in vivo dynamics of transcription may be applicable at a genome-wide scale and provide valuable insight into the dynamics of prokaryotic genetic networks.

We measured the in vivo production of RNA molecules tagged with MS2d-GFP in Escherichia coli, driven by the lar promoter, under weak and medium induction. The distributions of intervals between consecutive productions of RNAs are found to be sub-exponential, and the process of RNA production is found to be sub-Poissonian. We discuss possible models of transcription initiation and, based on our results and previous in vitro measurements, find that a sequential two-step model of transcription initiation at the promoter region explains well the results.

a b s t r a c tThe kinetics of transcription initiation in Escherichia coli depend on the duration of two rate-limiting steps, the closed and the open complex formation. In a lac promoter variant, P lac/ara-1 , the kinetics of these steps is controlled by IPTG and arabinose. From in vivo single-RNA measurements, we find that induction affects the mean and normalized variance of the intervals between consecutive RNA productions. Transcript production is sub-Poissonian in all conditions tested. The kinetics of each step is independently controlled by a different inducer. We conclude that the regulatory mechanism of P lac/ara-1 allows the stochasticity of gene expression to be environment-dependent.

Using a single-RNA detection technique in live Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG. Importantly, the distribution of waiting times of Plac/ara-1 is indistinguishable from that of PBAD, if and only if induced by arabinose alone. Finally, RNA production under the control of PBAD is found to be a sub-Poissonian process. We conclude that inducer-dependent waiting times affect mean and cell-to-cell diversity in RNA numbers long after induction, suggesting that intake mechanisms have non-negligible effects on the phenotypic diversity of cell populations in natural, fluctuating environments.

Programmed cell death is considered a key player in a variety of cellular processes that helps to regulate tissue growth, embryogenesis, cell turnover, immune response, and other biological processes. Among different types of cell death, apoptosis has been studied widely, especially in the field of cancer research to understand and analyse cellular mechanisms, and signaling pathways that control cell cycle arrest. Hallmarks of different types of cell death have been identified by following the patterns and events through microscopy. Identified biomarkers have also supported drug development to induce cell death in cancerous cells. There are various serological and microscopic techniques with advantages and limitations, that are available and are being utilized to detect and study the mechanism of cell death. The complexity of the mechanism and difficulties in distinguishing among different types of programmed cell death make it challenging to carry out the interventions and delay its progression. In this review, mechanisms of different forms of programmed cell death along with their conventional and unconventional methods of detection of have been critically reviewed systematically and categorized on the basis of morphological hallmarks and biomarkers to understand the principle, mechanism, application, advantages and disadvantages of each method. Furthermore, a very comprehensive comparative analysis has been drawn to highlight the most efficient and effective methods of detection of programmed cell death, helping researchers to make a reliable and prudent selection among the available methods of cell death assay. Conclusively, how programmed cell death detection methods can be improved and can provide information about distinctive stages of cell death detection have been discussed.

No abstract

No abstract