Interactions of the Non-coding RNA DsrA and RpoS mRNA with the 30 S Ribosomal Subunit

Worhunsky, David J.; Godek, Kristina; Litsch, Sarah; Schlax, Paula J.

doi:10.1074/jbc.m301684200

Cited by 18 publications

(20 citation statements)

References 39 publications

(79 reference statements)

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“…A recent in vivo analysis [124] suggested the following mechanistic model: Hfq associates with the helicase CsdA and binds to an A-rich sequence in the leader of rpoS, allowing the annealing of DsrA to rpoS and efficient translation. Nevertheless, Hfq binding does not seem to be necessary for the interaction of rpoS and DsrA with 30S [125]. The same group showed using photochemical crosslinking that the r-protein S1 interacts with both RNAs on the 30S [126] and suggested that S1 might play a role in facilitating translation of sRNA regulated mRNAs.…”

Section: Dsra/rpos Translation Activationmentioning

confidence: 89%

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics

et al. 2015

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Section: Dsra/rpos Translation Activationmentioning

confidence: 89%

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics

et al. 2015

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“…The 30S ribosomal subunit was purified from E. coli as described previously (41). lig RNA transcripts were synthesized with T7 RNA polymerase from PCR products by using procedures described previously (35). The template for the PCR was plasmid pRAT659, which harbored a lig=-=bglB fusion.…”

Section: Methodsmentioning

confidence: 99%

“…The structure of RNA can be influenced by ionic concentrations. For example, the accessibility of the RBS of the E. coli rpoS transcript is sensitive to magnesium concentration changes in vitro, indicating that conformational changes in the mRNA leader may facilitate osmotic regulation of rpoS (35). Additionally, the melting point of the Salmonella fourU thermometer is altered by shifts in magnesium concentrations within a physiological range (36).…”

mentioning

confidence: 99%

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

2013

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fThe spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5= untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in doublestranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA fMet -mRNA ternary complex was inhibited unless a 5= deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5= UTR and the first six codons were inserted between the Escherichia coli L-arabinose promoter and bgaB (␤-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the ␤-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5= UTR slightly diminished expression at each temperature tested. Finally, the expression level of ␤-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5= UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

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“…The procedure was carried out with cells grown to early stationary phase or with cells exposed to human serum (see Methods) to mimic systemic PAO1 infection (Van Delden, 2004). The protocol used here deviates from the coimmunoprecipitation of sRNA with Hfq antibodies from cell lysates (Zhang et al, 2003), and is biased against sRNAs that eventually associate with ribosomes (Worhunsky et al, 2003).…”

Section: Identification Of Ncrnas and Srna Candidates By Rnomicsmentioning

confidence: 99%

Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools

et al. 2008

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Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.

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Interactions of the Non-coding RNA DsrA and RpoS mRNA with the 30 S Ribosomal Subunit

Cited by 18 publications

References 39 publications

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools

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