Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant.

Hajnsdorf, Eliane; Steier, O.; Coscoy, Laurent; Teysset, Laure; Régnier, Philippe

doi:10.1002/j.1460-2075.1994.tb06639.x

Cited by 109 publications

(139 citation statements)

References 73 publications

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“…Although RNase R and PNPase are not known to work together, our data suggests that these enzymes may cooperate to convert the ϩ19 cleavage product to the ϩ13 product. Finally, RNase II can indirectly inhibit the degradation of structured mRNAs by removing 3Ј single-stranded regions required by PNPase to bind substrate (39,40). These biochemical properties are consistent with the accumulation of cleavage products in our exoribonuclease knock-out strains.…”

Section: Discussionsupporting

confidence: 84%

Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Garza-Sánchez¹,

Janssen²,

Hayes

2006

Journal of Biological Chemistry

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Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA 3 Gly in the peptidyl-tRNA binding site, and prolyl-tRNA 2 Pro is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA 2 Pro on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA 2 Pro readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses. A-site3 mRNA cleavage is a novel RNase activity that acts on A-site codons within paused ribosomes. Ehrenberg, Gerdes and their colleagues (1) first demonstrated that Escherichia coli RelE protein causes cleavage of A-site mRNA in vitro. Subsequently, A-site cleavage was also shown to occur at stop codons during inefficient translation termination in cells that lack RelE and related proteins (2, 3). The latter finding indicates that another unknown A-site nuclease also exists in E. coli. Indeed, it is possible the ribosome itself catalyzes A-site cleavage. The molecular requirements for A-site cleavage are incompletely understood, but an unoccupied ribosome A site appears to be important for both RelE-dependent and RelE-independent nuclease activity (1, 2).A-site nuclease activity truncates mRNAs and produces stalled ribosomes that are unable to continue standard translation. In bacteria, ribosomes stalled at the 3Ј termini of such truncated messages are "rescued" by the tmRNA quality control system. tmRNA is a specialized RNA that acts first as a tRNA to bind the A-site of stalled ribosomes, and then as an mRNA to direct the addition of the ssrA peptide degradation tag to the C terminus of the nascent polypeptide (4, 5). As a result of tmRNA activity, incompletely synthesized proteins are targeted for proteolysis and stalled ribosomes undergo normal translation termination and recycling (5). In this manner, A-site mRNA cleavage and tmRNA work together as a translational quality control system that responds to paused and stall...

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Section: Discussionsupporting

confidence: 84%

Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Garza-Sánchez¹,

Janssen²,

Hayes

2006

Journal of Biological Chemistry

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“…Ten micrograms of total RNA were separated either on 1% agarose formaldehyde gel or 6% polyacrylamide gels for mRNA and sRNA analysis, respectively, and analyzed by Northern blotting (Hajnsdorf et al 1994;Hajnsdorf and Régnier 1999). Templates for the synthesis of ArcZ, GcvB, SgrS, and oppA and ptsG RNA probes were obtained by PCR amplification using a oligonucleotide containing the T7 promoter and a RNA-specific oligonucleotide (indicated with prefix m) described in Supplemental Table S2.…”

Section: Rna Preparation Analysis and Labelingmentioning

confidence: 99%

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

Caillet

Gracia

Fontaine

et al. 2014

RNA

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A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.

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“…This indicated that the possible pairing of SraG-S or SraG to rpsO and rpsO-pnp transcripts did not affect the RNase E cleavage of the mRNA upstream of the terminator T1 (Fig. 1C), which is the limiting step in the degradation of the rpsO transcript (Régnier and Hajnsdorf 1991;Hajnsdorf et al 1994b). In addition, asRNA overproduction did not significantly affect RNase III cleavage efficiency at the RIII1 site (Figs.…”

Section: Srag Srna Participates In Pnpase Homeostasismentioning

confidence: 92%

“…The levels of the rpsO transcript were lower in the rnc mutant. This is presumably because the level of PNPase was higher in the rnc strain and because PNPase was responsible for the poly(A)-dependent degradation of the rpsO transcript (Hajnsdorf et al 1994b;Robert-Le Meur and Portier 1994). This decrease was partly rescued by overproduction of either SraG or SraG-S, suggesting that they could negatively control the level of PNPase.…”

Section: Srag Srna Participates In Pnpase Homeostasismentioning

confidence: 99%

The small RNA SraG participates in PNPase homeostasis

Fontaine

Gasiorowski

Gracia

et al. 2016

RNA

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The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3 ′ -5 ′ exoribonuclease involved in mRNA decay in Escherichia coli. The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp. Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.

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Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant.

Cited by 109 publications

References 73 publications

Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

The small RNA SraG participates in PNPase homeostasis

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