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Transcription profiling of E. coli cells overexpressing GraL for short periods of time

ABSTRACT: While studying greA expression, we noted presence of an intrinsic terminator in the leader region of greA mRNA transcript. We found this terminator to be quite efficient both in vivo and in vitro. This region seems to be conserved among many enteric bacteria. Judging from fitness experiments, the resulting short RNAs (50-59nt long) exert biological effects. This lead us to propose that greA leader region encodes a novel small non-coding RNA that we collectively call GraL. We used microarrays to detail the effects of GraL after short and longterm overproduction of GraL. Experiment Overall Design: E. coli cells harbouring plasmids overexpressing GraL or vector control, were monitored in an effort to elucidate a longterm (constitutive overexpression) or short term (15 min induction with IPTG) effects. This was done in two backgrounds- either in wt E. coli cells, or ppGpp0 cells (delta relA, delta spoT).

ORGANISM(S): Escherichia coli

SUBMITTER: Katarzyna Potrykus

PROVIDER: E-GEOD-15404 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcription profiling of E. coli cells overexpressing GraL for long periods of time

Project description:While studying greA expression, we noted presence of an intrinsic terminator in the leader region of greA mRNA transcript. We found this terminator to be quite efficient both in vivo and in vitro. This region seems to be conserved among many enteric bacteria. Judging from fitness experiments, the resulting short RNAs (50-59nt long) exert biological effects. This lead us to propose that greA leader region encodes a novel small non-coding RNA that we collectively call GraL. We used microarrays to detail the effects of GraL after short and longterm overproduction of GraL. Experiment Overall Design: E. coli cells harbouring plasmids overexpressing GraL or vector control, were monitored in an effort to elucidate a longterm (constitutive overexpression) or short term (15 min induction with IPTG) effects. This was done in two backgrounds- either in wt E. coli cells, or ppGpp0 cells (delta relA, delta spoT).

2009-12-19 | E-GEOD-15405 | biostudies-arrayexpress

Expression data from E. coli cells overexpressing GraL for long periods of time

2009-12-09 | GSE15405 | GEO

Expression data from E. coli cells overexpressing GraL for short periods of time

2009-12-09 | GSE15404 | GEO

Expression data from E. coli cells overexpressing either GreA or GreB in ppGpp0 cells in the dksA+ or dksA- background

Project description:Strains devoid of ppGpp (ΔrelA ΔspoT; called ppGpp0), and ppGpp0 dksA- exhibit several amino acid requirements for growth on minimal media. We found that overexpression of DksA can complement some of those requirements. Since DksA is a factor that binds to the RNA polymerase secondary channel, we wondered if other secondary channel proteins might also exert a similar role with respect to growth on minimal media. In our study we found that GreA and partially GreB can in fact complement these requirements under certain conditions. Here, we wished to investigate a broader effect of GreA and GreB on ppGpp0 and ppGpp0 dksA- strains. Since the parent strains are unable to grow in minimal media, we had to supplement the M9 glucose medium with a set of amino acids (DFHILQSTV). We found that both, GreA and GreB can affect a much larger set of genes in the absence of dksA, than in its presence. Also, GreA seems to affect more genes than GreB, under both conditions. We used microarrays to detail the effects of overproducing either GreA or GreB in cells devoid of ppGpp, in the dksA+ or dksA- background E. coli cells harbouring plasmids carrying either greA (pA = pHM1873) or greB (pB = pHM1874) genes, or vector control (pGB2), were monitored in an effort to elucidate the effects of their protein products. This was done in two backgrounds- either in E. coli ppGpp0 cells (ΔrelA, ΔspoT) carrying wt dksA gene or a dksA deletion.

2011-10-26 | E-GEOD-28795 | biostudies-arrayexpress

Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: Unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay

Project description:In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here, we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is a target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. Corresponding variations in protein levels led us to hypothesize that coordinated RNA decay represents another level in the hierarchical methionine biosynthesis control network to adjust methionine biosynthesis enzyme amounts to current metabolic requirements.

2021-08-10 | PXD020619 | Pride

Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: Unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay

2021-02-18 | PXD022677 | Pride

Disributions of RpoC SigA NusA and GreA in Bacillus subtilis

Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation, and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.

2011-04-10 | E-MEXP-3056 | biostudies-arrayexpress

Genome-wide transcriptional profiling of Bacillus subtilis greA deletion and GreA-D44A mutant strains

Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Additionally, we performed transcriptome comparison between in wild type and greA deletion and greA D44A mutant cells to see the effect of RNAP pausing to the transcriptomes. Our results indicate that inactivation of GreA has a limited impact on the transcriptome, and these effects are not directly related to RNAP accumulation in the promoter or promoter-proximal regions.

2011-04-10 | E-MEXP-3055 | biostudies-arrayexpress

Ultra-deep RNA-seq of in vitro and in vivo transcripts by Escherichia coli RNA polymerase

Project description:We report a high-resolution Illumina RNA-seq method that can analyze non-coded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. The RNA samples were generated by transcription of pPR9 plasmid that contains a 5.7 kb fragment of E. coli rpoBC operon transcribed from a strong lambda phage PR promoter and terminated at an fd phage transcription terminator. The reference transcription reaction was performed in a buffer with 5 mM MgCl2 to determine the standard error rate (barcode 1). To reduce fidelity, we replaced Mg2+ with Mn2+ (barcode 2). To increase fidelity, we added GreA/GreB proteins for proofreading activity (barode 3 and 4). The same transcrit was purified from E. coli cells (barcode 5).

2013-08-01 | E-GEOD-46479 | biostudies-arrayexpress

Understanding the roles of dksA and greA in inorganic polyphosphate accumulation in Escherichia coli

Project description:Inorganic polyphosphate (polyP) is synthesized by bacteria in response to various stresses, but the mechanism of its regulation is unknown. Mutants of Escherichia coli lacking the RNA polymerase-binding transcription factor dksA are defective in polyP synthesis after a nutrient limitation stress, and this defect is reversed in a dksA greA mutant. In this work, we used RNA sequencing to compare transcription in wild-type, dksA, and dksA greA strains of E. coli before and after nutrient limitation, to identify genes whose expression pattern correlates with ability to synthesize polyP.

2020-03-11 | GSE144816 | GEO

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