Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance.

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components.

Bacterial RNA chaperones and chaperone-like riboregulators: behind the scenes of RNA-mediated regulation of cellular metabolism.

In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences.

Impact of pseudouridylation, substrate fold, and degradosome organization on the endonuclease activity of RNase E.

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle.

RNA lifetime control, from stereochemistry to gene expression.

Through the activities of various multi-component assemblies, protein-coding transcripts can be chaperoned toward protein synthesis or nudged into a funnel of rapid destruction. The capacity of these machine-like assemblies to tune RNA lifetime underpins the harmony of gene expression in all cells. Some of the molecular machines that mediate transcript turnover also contribute to on-the-fly surveillance of aberrant mRNAs and non-coding RNAs.

Stem-loops direct precise processing of 3' UTR-derived small RNA MicL.

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing.

Substrate Recognition and Autoinhibition in the Central Ribonuclease RNase E.

The endoribonuclease RNase E is a principal factor in RNA turnover and processing that helps to exercise fine control of gene expression in bacteria. While its catalytic activity can be strongly influenced by the chemical identity of the 5' end of RNA substrates, the enzyme can also cleave numerous substrates irrespective of the chemistry of their 5' ends through a mechanism that has remained largely unexplained. We report structural and functional data illuminating details of both operational modes.

Activity of Vsr endonucleases encoded by Neisseria gonorrhoeae FA1090 is influenced by MutL and MutS proteins.

Background: The functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins.

RNase E and the High-Fidelity Orchestration of RNA Metabolism.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced.

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured.

Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing.

The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes.

In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3' ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease.

Recognition of the small regulatory RNA RydC by the bacterial Hfq protein.

Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators.

Licensing and due process in the turnover of bacterial RNA.

RNA enables the material interpretation of genetic information through time and in space. The creation, destruction and activity of RNA must be well controlled and tightly synchronized with numerous cellular processes. We discuss here the pathways and mechanism of bacterial RNA turnover, and describe how RNA itself modulates these processes as part of decision-making networks.

The social fabric of the RNA degradosome.

Bacterial transcripts each have a characteristic half-life, suggesting that the processes of RNA degradation work in an active and selective manner. Moreover, the processes are well controlled, thereby ensuring that degradation is orderly and coordinated. Throughout much of the bacterial kingdom, RNA degradation processes originate through the actions of assemblies of key RNA enzymes, known as RNA degradosomes.

The seed region of a small RNA drives the controlled destruction of the target mRNA by the endoribonuclease RNase E.

Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5'-end of the cognate-pairing "seed.

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.