The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis ...was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.
Post-transcriptional regulatory mechanisms are widespread in bacteria. Interestingly, current published data hint that some of these mechanisms may be non-random with respect to their phylogenetic ...distribution. Although small, trans-acting regulatory RNAs commonly occur in bacterial genomes, they have been better characterized in Gram-negative bacteria, leaving the impression that they may be less important for Firmicutes. It has been presumed that Gram-positive bacteria, in particular the Firmicutes, are likely to utilize cis-acting regulatory RNAs located within the 5' mRNA leader region more often than trans-acting regulatory RNAs. In this analysis we catalog, by a deep sequencing-based approach, both classes of regulatory RNA candidates for Bacillus subtilis, the model microorganism for Firmicutes. We successfully recover most of the known small RNA regulators while also identifying a greater number of new candidate RNAs. We anticipate these data to be a broadly useful resource for analysis of post-transcriptional regulatory strategies in B. subtilis and other Firmicutes.
Nano-RNases: oligo- or dinucleases? Lee, Vincent T; Sondermann, Holger; Winkler, Wade C
FEMS microbiology reviews,
11/2022, Letnik:
46, Številka:
6
Journal Article
Recenzirano
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Abstract
Diribonucleotides arise from two sources: turnover of RNA transcripts (rRNA, tRNA, mRNA, and others) and linearization of cyclic-di-nucleotide signaling molecules. In both cases, there ...appears to be a requirement for a dedicated set of enzymes that will cleave these diribonucleotides into mononucleotides. The first enzyme discovered to mediate this activity is oligoribonuclease (Orn) from Escherichia coli. In addition to being the enzyme that cleaves dinucleotides and potentially other short oligoribonucleotides, Orn is also the only known exoribonuclease enzyme that is essential for E. coli, suggesting that removal of the shortest RNAs is an essential cellular function. Organisms naturally lacking the orn gene encode other nanoRNases (nrn) that can complement the conditional E. coli orn mutant. This review covers the history and recent advances in our understanding of these enzymes and their substrates. In particular, we focus on (i) the sources of diribonucleotides; (ii) the discovery of exoribonucleases; (iii) the structural features of Orn, NrnA/NrnB, and NrnC; (iv) the enzymatic activity of these enzymes against diribonucleotides versus other substrates; (v) the known physiological consequences of accumulation of linear dinucleotides; and (vi) outstanding biological questions for diribonucleotides and diribonucleases.
Specialized enzymes, including oligoribonuclease (orn), nanornase A (NrnA), NrnB, and NrnC, share structural features that allow them to cleave dinucleotides that arise from RNA turnover and cyclic-di-nucleotide signaling.
Riboswitches are structured domains that usually reside in the noncoding regions of mRNAs, where they bind metabolites and control gene expression. Like their protein counterparts, these RNA gene ...control elements form highly specific binding pockets for the target metabolite and undergo allosteric changes in structure. Numerous classes of riboswitches are present in bacteria and they comprise a common and robust metabolite-sensing system.
Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular ...responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory ("metalloregulatory") proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni(2+) and Co(2+). These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co(2+)-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.
The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl ...cobalamine (AdoCbl)–responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding–response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.
Organisms maintain the correct balance of intracellular metals primarily through metal-sensing proteins that control transport and storage of the target ion(s). Here, we reveal the basis of metal ...sensing and genetic control by a metalloregulatory RNA. Our data demonstrate that a previously uncharacterized orphan riboswitch, renamed the “M-box,” is a divalent metal-sensing RNA involved in Mg
2+ homeostasis. A combination of genetic, biochemical, and biophysical techniques demonstrate that Mg
2+ induces a compacted tertiary architecture for M-box RNAs that regulates the accessibility of nucleotides involved in genetic control. Molecular details are provided by crystallographic structure determination of a Mg
2+-bound M-box RNA. Given the distribution of this RNA element, it may constitute a common mode for bacterial metal ion regulation, and its discovery suggests the possibility of additional RNA-based metal sensors in modern and primordial organisms.
Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular ...responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory (“metalloregulatory”) proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni2+ and Co2+. These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co2+-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.
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•NiCo RNA is the first conserved riboswitch class that responds to transition metals•NiCo selectively recognizes cobalt or nickel, binding with positive cooperativity•NiCo riboswitches control metal homeostasis by regulating metal transport proteins
Metal ions serve many essential roles but are toxic in excess; therefore, organisms utilize proteins that allosterically couple metal-sensing sites to control of gene expression. Furukowa et al. discover that certain noncoding RNAs can also perform this task, suggesting that such metalloregulation is not limited to proteins.
The RFN element is a highly conserved domain that is found frequently in the 5′-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport ...proteins. We report that this domain serves as the receptor for a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. Our results also indicate that in Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination of the rib-DEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA. Sequence and structural analyses indicate that the RFN element is a natural FMN-binding aptamer, the allosteric character of which is harnessed to control gene expression.
Abstract
All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2–5 nucleotides in length. These short RNA fragments are processed to ...monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5′ terminus of RNA substrates, NrnB acts at the 3′ terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3′-5′ exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.
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