Increased
            <i>rrn</i>
            Gene Dosage Causes Intermittent Transcription of rRNA in
            <i>Escherichia coli</i>

Voulgaris, Justina; French, Sarah L.; Gourse, Richard L.; Squires, Catherine L.

doi:10.1128/jb.181.14.4170-4175.1999

Cited by 31 publications

(72 citation statements)

References 22 publications

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“…The use of ectopic expression has been an extremely valuable tool for dissecting complex biological systems in a large number of eukaryotic organisms. While it has only rarely been used in prokaryotes (Singer and Kaiser, 1995;Voulgaris et al, 1999), when properly utilized, it provides a powerful method for determining the impact of an individual protein in a particular pathway. Here, we have used an IPTG-regulated pcnB gene to examine the role of PAP I in the post-transcriptional regulation of RNA metabolism in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

Mohanty

Kushner

1999

Molecular Microbiology

131

260

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SummaryTo help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased halflives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Longterm overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3Ј-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.

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Section: Discussionmentioning

confidence: 99%

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

Mohanty

Kushner

1999

Molecular Microbiology

131

260

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“…The limitation of RNAP for genome-wide transcription is exacerbated in a fast-growing cell. It is estimated that the rate of rRNA synthesis approaches one initiation per second per rrn operon in a cell under optimal growth conditions (Bremer & Dennis, 1996;Voulgaris et al, 1999). The long length of the rrn transcripts coupled with the gene dosage effect described above has clear implications for the distribution of RNAP in the genome in response to cell growth.…”

Section: The Distribution Of Rnap and The Regulation Of Rrna Synthesismentioning

confidence: 99%

Growth rate regulation inEscherichia coli

2012

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Growth rate regulation in bacteria has been an important issue in bacterial physiology for the past 50 years. This review, using Escherichia coli as a paradigm, summarizes the mechanisms for the regulation of rRNA synthesis in the context of systems biology, particularly, in the context of genome-wide competition for limited RNA polymerase (RNAP) in the cell under different growth conditions including nutrient starvation. The specific location of the seven rrn operons in the chromosome and the unique properties of the rrn promoters contribute to growth rate regulation. The length of the rrn transcripts, coupled with gene dosage effects, influence the distribution of RNAP on the chromosome in response to growth rate. Regulation of rRNA synthesis depends on multiple factors that affect the structure of the nucleoid and the allocation of RNAP for global gene expression. The magic spot ppGpp, which acts with DksA synergistically, is a key effector in both the growth rate regulation and the stringent response induced by nutrient starvation, mainly because the ppGpp level changes in response to environmental cues. It regulates rRNA synthesis via a cascade of events including both transcription initiation and elongation, and can be explained by an RNAP redistribution (allocation) model.

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“…Such strategies have been used in simulation by Klump et al [39] to estimate the pause duration in presence/absence of anti-termination complex for ribosomal genes. Other model parameters (RNAP footprint, gene-length, and hopping rate) are directly taken from experimental measurements [11,51] and are kept constant. The fitted values for the control strain of each dataset are listed in Table 2.…”

Section: Analysis Of Rna Polymerase Number Distributions For Ribosomamentioning

confidence: 99%

“…Recent experimental advancements have stimulated the analysis of the mechanisms of transcription elongation in vivo. Electron micrograph (EM) images [10][11][12][13] enable counting nascent RNAs i.e. the number of RNAP molecules engaged in the elongation process of any one gene (shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

Probing mechanisms of transcription elongation through cell-to-cell variability of RNA polymerase

Ali

Choubey

Das

et al. 2019

Preprint

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The process of transcription initiation and elongation are primary points of control in the regulation of gene expression. While biochemical studies have uncovered the mechanisms involved in controlling transcription at each step, how these mechanisms manifest in vivo at the level of individual genes is still unclear. Recent experimental advances have enabled single-cell measurements of RNAP molecules engaged in the process of transcribing a gene of interest. In this manuscript, we use Gillespie simulations to show that measurements of cellto-cell variability of RNAP numbers and inter-polymerase distances can reveal the prevailing mode of regulation of a given gene. Mechanisms of regulation at each step, from initiation to elongation dynamics, produce qualitatively distinct signatures which can further be used to discern between them. Intriguingly, depending on the initiation kinetics, stochastic elongation can either enhance or suppress cell-to-cell variability at the RNAP level. To demonstrate the value of this framework, we analyze RNAP number distribution data for ribosomal genes in S. cerevisiae from three previously published studies and show that this approach provides crucial mechanistic insights into the transcriptional regulation of these genes. Author SummaryThe process of transcription comprises many distinct steps and understanding the regulation of each of these steps provides insight into how the levels of gene expression are controlled in the cell. In this manuscript, we use stochastic simulations to explore how regulation at the level of elongation and initiation together influences the distribution of actively transcribing RNAP on a gene. We find that each of these steps of regulation leaves a distinct imprint on the gene-to-gene variability of number of actively transcribing RNAP on the gene and their inter-RNAP distances. Using these results, we analyze recent experimental data of transcribing RNAP distributions and find that the perturbations in these studies primarily impact the transcription initiation dynamics of the genes.

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Increased rrn Gene Dosage Causes Intermittent Transcription of rRNA in Escherichia coli

Cited by 31 publications

References 22 publications

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

Growth rate regulation inEscherichia coli

Probing mechanisms of transcription elongation through cell-to-cell variability of RNA polymerase

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