Fluctuations in spo0A Transcription Control Rare Developmental Transitions in Bacillus subtilis

Phosphorylated Spo0A is a master regulator of stationary phase development in the model bacterium Bacillus subtilis, controlling the formation of spores, biofilms, and cells competent for transformation. We have monitored the rate of transcription of the spo0A gene during growth in sporulation medium using promoter fusions to firefly luciferase. This rate increases sharply during transient diauxie-like pauses in growth rate and then declines as growth resumes. In contrast, the rate of transcription of an rRNA gene decreases and increases in parallel with the growth rate, as expected for stable RNA synthesis. The growth pause-dependent bursts of spo0A transcription, which reflect the activity of the spo0A vegetative promoter, are largely independent of all known regulators of spo0A transcription. Evidence is offered in support of a “passive regulation” model in which RNA polymerase stops transcribing rRNA genes during growth pauses, thus becoming available for the transcription of spo0A. We show that the bursts are followed by the production of phosphorylated Spo0A, and we propose that they represent initial responses to stress that bring the average cell closer to the thresholds for transition to bimodally expressed developmental responses. Measurement of the numbers of cells expressing a competence marker before and after the bursts supports this hypothesis. In the absence of ppGpp, the increase in spo0A transcription that accompanies the entrance to stationary phase is delayed and sporulation is markedly diminished. In spite of this, our data contradicts the hypothesis that sporulation is initiated when a ppGpp-induced depression of the GTP pool relieves repression by CodY. We suggest that, while the programmed induction of sporulation that occurs in stationary phase is apparently provoked by increased flux through the phosphorelay, bet-hedging stochastic transitions to at least competence are induced by bursts in transcription

Analysis of the determinants of Pol II pausing

Pausing of transcribing RNA polymerase II (Pol II) has emerged as a general feature of gene expression in human cells. Many transcription factors, DNA sequences and chromatin characteristics have been implicated in inducing transcriptional pausing. However, it is unclear what are the relative contributions of these factors on the observed Pol II pausing. Furthermore, research in metazoans has mainly focused on Pol II promoter-proximal pausing, leaving the causes of pausing outside of this region unknown. To reliably detect real transcriptional pausing sites and advance the understanding of the causes of this phenomenon, we developed a pausing detection algorithm for nucleotide-resolution Pol II occupancy data. We scrutinized the characteristics and potential shortcomings of Native Elongating Transcript sequencing (NET-seq), which is one of the high-resolution methods of Pol II profiling, and we used our observations to improve the NET-seq processing pipeline. Leveraging the improved processing pipeline and the developed pausing detection algorithm revealed widespread genome-wide Pol II pausing at a nucleotide resolution in human cells. Next, we set out to identify the determinants of Pol II pausing in an unbiased manner based on the underlying DNA sequence. To predict the predisposition of a genomic site to evoke Pol II pausing, we applied a range of machine learning approaches using previously identified high-confidence pausing sites. For each of the sites, we created a large number of features, including both factors that were previously linked to transcriptional pausing and factors that were not yet implicated in invoking pausing. Our analysis revealed DNA sequence properties underlying widespread Pol II pausing including a new pausing motif. Interestingly, key sequence determinants of RNA polymerase pausing are shared by human cells and bacteria. Our study indicates that transcriptional pausing in human cells is sequence-induced and that the determinants of Pol II pausing might be evolutionary conserved.Ein allgemeines Merkmal der Genexpression in menschlichen Zellen ist das Pausieren der RNA Polymerase II (Pol II). Verschiedene Aspekte wie Transkriptionsfaktoren, DNA Sequenzen und Eigenschaften des Chromatins werden mit dem Prozess in Verbindung gebracht. Der relative Beitrag dieser Faktoren zur Entstehung der beobachteten Pausen ist unbekannt. Darüber hinaus hat sich die bisherige Forschung bei Metazoen hauptsächlich auf Pol II Pausen während der frühen Elongationsphase, im promoter-proximalen Bereich, konzentriert. Die Ursachen für das Pausieren außerhalb dieser Regionen sind unbekannt. Um das Verständnis der Ursachen von Transkriptionspausen zu verbessern, haben wir einen Algorithmus entwickelt, der Pol II Signale verarbeitet und Pausen präzise bis auf ein einzelnes Nukleotid lokalisiert. Die Pol II Signalmessungen werden mithilfe von NET-seq (Native Elongating Transcript Sequencing), einer hochauflösenden Methode, erstellt. Bei der Untersuchung der Methode identifizierten wir systematische Fehler in den Messdaten, welche zur Anpassung bei der Datenverarbeitung führte. Diese algorithmischen Verbesserungen zeigten, dass Pol II Pausen in menschlichen Zellen weit verbreitet sind und verteilt über das gesamte Genom, an einzelnen Nukleotiden, beobachtet werden können. Für eine unvoreingenommene Identifizierung der Sequenzspezifischen Faktoren, die zum Pausieren der Pol II beitragen, wurden eine Reihe von Methoden des maschinellen Lernens angewandt. Mit hoher Sicherheit detektierte Transkriptionspausen wurden genutzt, um Prädispositionen in DNA-Abschnitten zu lernen und vorherzusagen. Für jedes dieser Beispiel Regionen werden beschreibende Merkmale erstellt. Darunter befinden sich Faktoren, die zuvor mit Transkriptionspausen in Verbindung gebracht wurden, sowie Merkmale ohne bekannte Assoziation. Unsere Analyse identifiziert ein neues DNA Sequenzmotiv und andere relevante Sequenzeigenschaften, welche dem pausieren der Pol II zugrunde liegen. Interessanterweise sind die identifizierten Sequenzeigenschaften sowohl in menschlichen Zellen als auch in Bakterien zu finden. Unsere Studie deutet darauf hin, dass Transkriptionspausen in menschlichen Zellen sequenzabhängig und evolutionär konserviert sind

Molecular Mechanisms Regulating Rna Polymerase Ii Pausing During Gene Activation

RNA Polymerase II (Pol II), the enzyme that transcribes all messenger RNAs (mRNAs) has another activity by pausing at gene promoters. The paused Pol II generates short 5’-capped RNAs of about 50 nucleotides in length. Much is known about Pol II pausing including its prevalence across the genome. But, the molecular mechanisms that are involved in regulating Pol II pausing and its roles in gene regulation are yet to be fully explored. In this study, I have investigated the molecular mechanisms that regulate dynamics of Pol II pausing in response to gene activation. The main goal of this study was to determine how cells respond to stimuli by altering Pol II pausing states. To this end, we characterized changes in Pol II pausing in MCF-7 human breast cancer cells, using two distinct stimuli, heat shock and Trichostatin A (TSA), a histone deacetylase inhibitor. Two genes SNAI1, a master regulator of epithelial to mesenchymal transition, and HSPA1B (heat shock protein 70), a master regulator of heat shock response, show mRNA upregulation upon treatment with both stimuli. To determine changes in Pol II pausing and its dynamics in response to gene activation, I have used permanganate footprinting for single gene analysis, and short-capped RNA sequencing (scRNA-seq) for genome-wide analysis. I have shown that, upon activation, paused genes can retain Pol II pausing and non-paused genes can acquire Pol II pausing. Further, I observe that genes such as HSPA1B, undergo pause release during heat shock activation but SNAI1 does not. In addition, I have shown that the turnover of the paused Pol II complex changes in a stimulus-specific manner, indicating that the release of paused Pol II is sensitive to the nature of the stimulus. To investigate the rate of turnover of the paused complex, I used a specific inhibitor of a general transcription factor TFIIH, Triptolide and measured the residence time of the Pol II complex at the paused site. Genome-wide analysis of Pol II turnover demonstrates that not all genes respond the same way to heat shock. This project describes a novel mechanism for regulation of transcription during gene activation in human cells during responses to environmental stresses

Cell-to-cell diversity in protein levels of a gene driven by a tetracycline inducible promoter

<p>Abstract</p> <p>Background</p> <p>Gene expression in <it>Escherichia coli </it>is regulated by several mechanisms. We measured in single cells the expression level of a single copy gene coding for green fluorescent protein (GFP), integrated into the genome and driven by a tetracycline inducible promoter, for varying induction strengths. Also, we measured the transcriptional activity of a tetracycline inducible promoter controlling the transcription of a RNA with 96 binding sites for MS2-GFP.</p> <p>Results</p> <p>The distribution of GFP levels in single cells is found to change significantly as induction reaches high levels, causing the Fano factor of the cells' protein levels to increase with mean level, beyond what would be expected from a Poisson-like process of RNA transcription. In agreement, the Fano factor of the cells' number of RNA molecules target for MS2-GFP follows a similar trend. The results provide evidence that the dynamics of the promoter complex formation, namely, the variability in its duration from one transcription event to the next, explains the change in the distribution of expression levels in the cell population with induction strength.</p> <p>Conclusions</p> <p>The results suggest that the open complex formation of the tetracycline inducible promoter, in the regime of strong induction, affects significantly the dynamics of RNA production due to the variability of its duration from one event to the next.</p

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing

Dynamics of Stochastic Sequence-Level Models of Transcription and Translation in Prokaryotes

In prokaryotes, transcription and translation are dynamically coupled, as the latter starts before the former is completed. Also, from one transcript, several translation events occur in parallel. To study how events in transcription elongation affect translation elongation and fluctuations in protein levels, we propose a delayed stochastic model of prokaryotic transcription and translation at the nucleotide and codon level that includes the promoter open complex formation and alternative pathways to elongation, namely pausing, arrests, editing, pyrophosphorolysis, RNA polymerase traffic, and premature termination. Stepwise translation can start after the ribosome binding site is formed and accounts for variable codon translation rates, ribosome traffic, back-translocation, drop-off, and trans-translation. The recent development of measurement techniques in genetics promises better un-derstanding of the functioning of biological systems. To attain the most out of these techniques, new methods are needed of interpreting the data, since most existent me-thods have been developed to analyze population level measurements, rather than ex-tracting information from single cell dynamics. For example, one needs accurate estima-tion of the measurement noise from single cell measurements of gene expression. We use recently developed methods to measure gene expression in vivo in individual cells, at the single RNA and protein molecule levels. Such measurements of gene expression, attained in various conditions, as well as the proposed modeling strategy, are used to study and model the dynamics of gene expression at the single event level and to esti-mate noise sources in the processes. First, the model is shown to accurately match the measurements of sequence-dependent translation elongation dynamics. Next, the degree of coupling between fluc-tuations in RNA and protein levels, and its dependence on the rates of transcription and translation initiation is characterized. Finally, sequence-specific transcriptional pauses are found to have an effect on protein noise levels. For parameter values within realistic intervals, transcription and translation are found to be tightly coupled in Escherichia coli, as the noise in protein levels is mostly determined by the underlying noise in RNA levels. Sequence-dependent events in transcription elongation, e.g. pauses, are found to cause tangible effects in the degree of fluctuations in protein levels, implying that these are evolvable. /Kir1

One step back before moving forward: regulation of transcription elongation by arrest and backtracking

RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.Ministerio de Economía y Competitividad de España. BFU2007-67575-C03-02 y BFU-2010-21975-C03-03Junta de Andalucía. P07-CVI-02623 y P08-CVI-0350

The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase