Search

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Common bean is a legume with high demand for human consumption and with high protein content on its seeds. The seed filling stage is a crucial step to obtain high-quality seeds with a good level of nutrients. For this, it is necessary for a correct communication between the different seed compartments. Nucleotides are essential components with nitrogen and phosphorous on its molecules, and its metabolism in seed development has not been studied in detail. In this manuscript, we have studied nucleotide metabolism in common bean pods during seed filling stage at pod valves, seed coats, and embryos. Nuclease and ribonuclease activities were assayed as nucleotide-generating enzymes, and nucleotidase, nucleosidase, and allantoinase as nucleotide-degrading activities. Nuclease was predominant in seed coats whereas ribonuclease was equally determined in seed coats and valves, although with differences in the three ribonucleases determined (16, 17, and 19 kDa). Nucleotidase and nucleosidase activities were detected in the three pods parts, and differently to nucleic degrading activities with significant activity in embryos. The relative expression of gene families coding for all these activities (S1 nuclease, S-like T2 ribonuclease, nucleotidase, nucleosidase and allantoinase) in the three pods parts was also studied. We have found the highest level of expression for some members of each family in seed coats. The allantoinase data suggest that nucleotide might be fully degraded in valves and seed coats but not in embryos. Overall, the data presented allow to conclude that there is an intense nucleotide metabolism in fruits during the seed filling stage with an especial involvement of seed coats in the process. [ABSTRACT FROM AUTHOR]

Recent cryo-electron microscopy (cryo-EM) structures have offered critical new insights into the molecular mechanisms governing tRNA biogenesis. Structural and functional studies along with new advances in tRNA sequencing have revealed new details on the processing of tRNAs into tRNA-derived fragments (tRFs) by RNase A family members, RNase L, and Dicer. Recent structural advances and reconstitution experiments have provided new insights into the mechanism of surveillance and decay of tRNAs by exoribonucleases, including the Xrn nucleases and the exosome. Ribonucleases (RNases) such as the SLFN family, ANKZF1, and SAMD9 have new links with tRNA quality control and cellular defense mechanisms. Defining the cellular cues and molecular mechanisms that regulate tRNA processing and decay will enhance our understanding of human diseases associated with tRNA dysregulation. Ribonucleases (RNases) play important roles in supporting canonical and non-canonical roles of tRNAs by catalyzing the cleavage of the tRNA phosphodiester backbone. Here, we highlight how recent advances in cryo-electron microscopy (cryo-EM), protein structure prediction, reconstitution experiments, tRNA sequencing, and other studies have revealed new insight into the nucleases that process tRNA. This represents a very diverse group of nucleases that utilize distinct mechanisms to recognize and cleave tRNA during different stages of a tRNA's life cycle including biogenesis, fragmentation, surveillance, and decay. In this review, we provide a synthesis of the structure, mechanism, regulation, and modes of tRNA recognition by tRNA nucleases, along with open questions for future investigation. [ABSTRACT FROM AUTHOR]

ABSTRACT To explore the influence of storage temperature and time on the stability of different concentrations of hepatitis C virus nucleic acid (HCV RNA) samples and to provide data reference for laboratory quality control. Serum samples of 10 patients with HCV RNA detection quantitation of 106–108 IU/mL were collected. The samples of each patient were diluted into three concentrations: high, medium, and low. Then the samples of each concentration were divided into 21, which were divided into three groups according to the storage conditions of −20°C, 4°C, and 25°C, with seven samples in each group. The samples were selected from each group for quantitative detection of HCV RNA on day 0, day 1, day 3, day 5, day 7, day 14, and day 30. The results of each concentration and storage temperature sample remained stable within 5 days. Based on the mixed-effect linear model, the main effects of temperature, time, and concentration were statistically significant (P < 0.01). There was an interaction effect between concentration and time (P = 0.0448), and there was also an interaction effect between temperature and time (P < 0.01). There was no interaction effect between concentration and temperature (P = 0.11) or between concentration, temperature, and time (P = 0.90). The results of serum samples with different concentrations of the HCV RNA remained stable within 5 days. The lower the initial concentration of HCV RNA serum sample, the worse the stability; the higher the storage temperature, the worse the stability. If conditions permit, the laboratory should store such samples at −20°C.IMPORTANCEPreviously, there were few reports about the influence of different concentrations of sample nucleic acid on the stability of samples at various temperatures and times in various literatures. Therefore, it is necessary to analyze the influence of concentration factors on the stability of samples and test results at different storage times and temperatures. This study took the concentration of hepatitis C virus nucleic acid as the research object to further understand the stability of hepatitis C virus nucleic acid test samples under various storage conditions, to provide data reference for the treatment of hepatitis C virus nucleic acid and RNA test samples before clinical laboratory test, and provide guidance and help for the improvement of laboratory quality control.

The dynamic behavior of Protein Disulfide Isomerase (PDI) in an aqueous solution environment under physiologically active pH has been experimentally verified in this study using Small Angle X-ray Scattering (SAXS) technique. The structural mechanism of dimerization for full-length PDI molecules and co-complex with two renowned substrates has been comprehensively discussed. The structure models obtained from the SAXS data of PDI purified from bovine liver display behavior duality between unaccompanied-enzyme and after engaged with substrates. The analysis of SAXS data revealed that PDI exists as a homo-dimer in the solution environment, and substrate induction provoked its segregation into monomer to enable the enzyme to interact systematically with incoming clients. [ABSTRACT FROM AUTHOR]

Summary: Plants activate immunity upon recognition of pathogen‐associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses.Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide‐ and catalytic residue‐dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles.Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern‐triggered immunity responses. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single‐stranded RNAs at guanosine, leaving a 3′‐end phosphate. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues.We propose that the pathogen‐derived apoplastic guanosine‐specific single‐stranded endoribonucleases lead to immunity potentiation in plants. [ABSTRACT FROM AUTHOR]

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID‐19 pandemic caused by the etiological agent SARS‐CoV‐2. All common assays for infection rely on the detection of viral sub‐components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID‐19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false‐negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS‐CoV‐2 main protease, 3CLpro. Zymogen activation by 3CLpro leads to a >300‐fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen‐detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications. [ABSTRACT FROM AUTHOR]

ABSTRACT Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus Colletotrichum fructicola, which causes pear anthracnose, can colonize different plant tissues like leaves and fruits, which are occupied by a diversity of microbes. We speculate that this fungus produces antimicrobial effectors to outcompete host-associated competitive microorganisms. Herein, we identified two secreted ribonucleases, CfRibo1 and CfRibo2, from the C. fructicola secretome. The two ribonucleases both possess ribonuclease activity and showed cytotoxicity in Nicotianan benthamiana without triggering immunity in an enzymatic activity-dependent manner. CfRibo1 and CfRibo2 recombinant proteins exhibited toxicity against Escherichia coli, Saccharomyces cerevisiae, and, importantly, the phyllosphere microorganisms isolated from the pear host. Among these isolated microbial strains, Bacillus altitudinis is a pathogenic bacterium causing pear soft rot. Strikingly, CfRibo1 and CfRibo2 were found to directly antagonize B. altitudinis to facilitate C. fructicola infection. More importantly, CfRibo1 and CfRibo2 functioned as essential virulence factors of C. fructicola in the presence of host-associated microorganisms. Further analysis revealed these two ribonucleases are widely distributed in fungi and are undergoing purifying selection. Our results provide the first evidence of antimicrobial effectors in Colletotrichum fungi and extend the functional diversity of fungal ribonucleases in plant-pest-environment interactions.IMPORTANCEColletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in various crops in agriculture, and understanding how this fungus establishes successful infection is of great significance for anthracnose disease management. Fungi are known to produce secreted effectors as weapons to promote virulence. Considerable progress has been made in elucidating how effectors manipulate plant immunity; however, their importance in modulating environmental microbes is frequently neglected. The present study identified two secreted ribonucleases, CfRibo1 and CfRibo2, as antimicrobial effectors of C. fructicola. These two proteins both possess toxicity to pear phyllosphere microorganisms, and they efficiently antagonize competitive microbes to facilitate the infection of pear hosts. This study represents the first evidence of antimicrobial effectors in Colletotrichum fungi, and we consider that CfRibo1 and CfRibo2 could be targeted for anthracnose disease management in diverse crops in the future.

A promising approach to help students safely return to in person learning is through the application of sentinel cards for accurate high resolution environmental monitoring of SARS-CoV-2 traces indoors. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface materials, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, Wet Wipes, RNase Away) at three different viral loads: "high" (4 × 104 GE/mL), "medium" (1 × 104 GE/mL), and "low" (2.5 × 103 GE/mL). RNase Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet Wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. IMPORTANCE Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are a persistent signal or new infectious cases and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

Bacteria and archaea respond and adapt to environmental stress conditions by modulating the toxin–antitoxin (TA) system for survival. Within the bacterium Helicobacter pylori, the protein HP0894 is a key player in the HP0894-HP0895 TA system, in which HP0894 serves as a toxin and HP0895 as an antitoxin. HP0894 has intrinsic ribonuclease (RNase) activity that regulates gene expression and translation, significantly influencing bacterial physiology and survival. This activity is influenced by the presence of metal ions such as Mg2+. In this study, we explore the metal-dependent RNase activity of HP0894. Surprisingly, all tested metal ions lead to a reduction in RNase activity, with zinc ions (Zn2+) causing the most significant decrease. The secondary structure of HP0894 remained largely unaffected by Zn2+ binding, whereas structural rigidity was notably increased, as revealed using CD analysis. NMR characterized the Zn2+ binding, implicating numerous His, Asp, and Glu residues in HP0894. In summary, these results suggest that metal ions play a regulatory role in the RNase activity of HP0894, contributing to maintaining the toxin molecule in an inactive state under normal conditions. [ABSTRACT FROM AUTHOR]

Objective: Among modern gene therapy methods for combating oncology, suicidal gene therapy based on the delivery of a cytotoxic agent to target cells is of particular importance and promise. As one of such genes, the gene for ribonuclease of Bacillus pumilus 7P, binase, can be considered; the enzyme has a high antitumor potential and low immunogenicity. In addition to the choice of a transgene, another factor influencing the effectiveness of gene therapy is the method of delivering the nucleic acid to target cells. Surfactants have high functional activity and are promising means of delivering therapeutic nucleic acids. The aim of this work was to evaluate the possibility of using geminal surfactants as a means of delivering a genetic construct based on the cytotoxic binase gene into tumor cells. Methods: To optimize the transfection conditions, a reporter genetic construct carrying the binase gene fused to the gene for the green fluorescent protein TurboGFP was created, which made it possible to evaluate the delivery efficiency by the fluorescence intensity. To eliminate the toxic effect of binase on recipient cells, the RNase inhibitor gene, barstar, was introduced into the genetic construct. Results and Discussion: A high complexing ability of geminal surfactants in relation to the reporter system was shown by methods of dynamic light scattering and fluorescence spectroscopy. For surfactant 16-6-16OH, the highest transfecting activity together with a low level of cytotoxicity was found. Conclusions: Thus, the study proved the possibility of using geminal surfactants for the delivery of therapeutic nucleic acids to target cells. [ABSTRACT FROM AUTHOR]

Although bacterial detection by 16S rRNA gene amplicon DNA sequencing is a widely-applied technique, standardized methods for sample preparation and DNA extraction are needed to ensure accuracy, reproducibility, and scalability for automation. To develop these methods for bovine bulk milk, we assembled and tested a bacterial cell mock community (BCMC) containing bacterial species commonly found in milk. The following protocol variations were examined:: BCMC enumeration (colony enumeration or microscopy), sample volume (200 μl to 30 ml), sample storage condition (frozen in PBS or 25% glycerol or exposure to freeze-thaw cycles), cell lysis method (bead-beating, vortex, enzymatic), and DNA extraction procedure (MagMAX Total, MagMAX CORE, and MagMAX Ultra 2.0, with and without either Proteinase K or RNase A). Cell enumeration by microscopy was more accurate for quantification of the BCMC contents. We found that least 10 mL (≥ 104 cells in high quality milk) is needed for reproducible bacterial detection by 16S rRNA gene amplicon DNA sequencing, whereas variations in storage conditions caused minor differences in the BCMC. For DNA extraction and purification, a mild lysis step (bead-beating for 10 s at 4 m/s or vortexing at 1800 rpm for 10 s) paired with the MagMAX Total kit and Proteinase K digestion provided the most accurate representation of the BCMC. Cell lysis procedures conferred the greatest changes to milk microbiota composition and these effects were confirmed to provide similar results for commercial milk samples. Overall, our systematic approach with the BCMC is broadly applicable to other milk, food, and environmental samples therefore recommended for improving accuracy of culture-independent, DNA sequence-based analyses of microbial composition in different habitats.

Abstract The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five $${\text{SO}}_{4}^{2 - }$$ SO 4 2 - moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851–861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cβ. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989) includes a sulphate anion which occupies approximately the same location as the $${\text{PO}}_{4}^{2 - }$$ PO 4 2 - phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743–755, 1983). The present structure contains 5 $${\text{SO}}_{4}^{2 - }$$ SO 4 2 - groups SO41151–SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201–EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989).

ABSTRACT Toxin-antitoxin (TA) systems are widespread among prokaryotes and can play an important role in the maintenance of genetic elements and pathogen virulence. Their roles in bacterial stress response and antimicrobial persistence currently remain highly controversial. The current study investigated a novel TA system (Aave_1720-Aave_1719) from the phytopathogenic bacterium Acidovorax citrulli, which had characteristics of both type II and type V TA systems. Heterologous expression in Escherichia coli confirmed that Aave_1720 functions as a toxin gene, while Aave_1719 acts as antitoxin gene. The Aave_1720 toxin functions as a ribonuclease, and antitoxin Aave_1719 binds directly to Aave_1720 to inhibit its ribonuclease activity, which is typical of type II TA systems. Interestingly, Aave_1719 itself was found to exhibit ribonuclease activity with a preference for targeting Aave_1720 mRNA, which indicates that it can also neutralize the Aave_1720 toxin in a manner characteristic of type V TA systems. Expression analysis revealed that the Aave_1719 gene is upregulated in response to sodium hypochlorite stress and that deletion of the Aave_1720 gene individually or in combination with Aave_1719 affected biofilm formation, as well as the survival of A. citrulli in response to sodium hypochlorite stress. Taken together, these findings expand our understanding of TA systems revealing a novel mechanism of toxin neutralization that combines aspects of both type II and type V TA systems. IMPORTANCE Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

T2 ribonucleases are endoribonucleases that are found in every organism and that carry out important biological functions. In plants, T2 ribonucleases are organized into multi-gene families, and each member is thought to have a specific function. In this study, the ribonuclease activity has been analyzed in common bean seedlings during germination and it was found that the activity was very high in the testa during this process. This high activity correlated with a high level of expression of the S-like ribonuclease T2 PvRNS3. The protein encoded by this gene was overexpressed in Escherichia coli and characterized. The purified protein showed ribonuclease activity with RNA and not with DNA, confirming that PvRNS3 encodes a ribonuclease. PvRNS3 is an acidic ribonuclease with remarkable heat stability, of which activity is inhibited by Cu and Zn, as well as by ditiotreitol (DTT). PvRNS3 expression was also selectively induced in some stress situations, such as salt stress in radicles and wounded leaves. The high level of expression in the testa and high ribonuclease activity suggest an additional role for the testa in common bean germination, apart from being a protective barrier for embryos in seeds. The putative role of this ribonuclease in the extracellular space after seed hydration and release to the surrounding space to improve seedling fitness is discussed. [ABSTRACT FROM AUTHOR]

Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis-based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity. [ABSTRACT FROM AUTHOR]

Abstract Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram‐negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli, feedback‐regulate RNase E expression via the rne 5′‐untranslated region (5′ UTR) in cis. However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full‐length rne mRNA caused by the stabilization of its 5′ UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3′ ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′ UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.

The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.

The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five SO 4 2 - moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851–861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cβ. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989) includes a sulphate anion which occupies approximately the same location as the PO 4 2 - phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743–755, 1983). The present structure contains 5 SO 4 2 - groups SO41151–SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201–EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989). [ABSTRACT FROM AUTHOR]

β-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B β-lactamases are members of a superfamily of metallo-β-lactamase (MβL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MβL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MβL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as β-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MβL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MβL fold enzymes with demonstrated enzymatic or non-enzymatic activities. [ABSTRACT FROM AUTHOR]

The instability of messenger RNA is crucial to the control of gene expression. In Bacillus subtilis, RNase Y is the major decay-initiating endoribonuclease. Here, we show how this key enzyme regulates its own synthesis by modulating the longevity of its mRNA. Autoregulation is achieved through cleavages in two regions of the rny (RNase Y) transcript: (i) within the first ~100 nucleotides of the open reading frame, immediately inactivating the mRNA for further rounds of translation; (ii) cleavages in the rny 5′ UTR, primarily within the 5′-terminal 50 nucleotides, creating entry sites for the 5′ exonuclease J1 whose progression is blocked around position −15 of the rny mRNA, potentially by initiating ribosomes. This links the functional inactivation of the transcript by RNase J1 to translation efficiency, depending on the ribosome occupancy at the translation initiation site. By these mechanisms, RNase Y can initiate degradation of its own mRNA when the enzyme is not occupied with degradation of other RNAs and thus prevent its overexpression beyond the needs of RNA metabolism. [ABSTRACT FROM AUTHOR]

Polyketide metabolism‐associated proteins in Mycobacterium tuberculosis play an essential role in the survival of the bacterium, which makes them potential drug targets for the treatment of tuberculosis (TB). The novel ribonuclease protein Rv1546 is predicted to be a member of the steroidogenic acute regulatory protein‐related lipid‐transfer (START) domain superfamily, which comprises bacterial polyketide aromatase/cyclases (ARO/CYCs). Here, we determined the crystal structure of Rv1546 in a V‐shaped dimer. The Rv1546 monomer consists of four α‐helices and seven antiparallel β‐strands. Interestingly, in the dimeric state, Rv1546 forms a helix‐grip fold, which is present in START domain proteins, via three‐dimensional domain swapping. Structural analysis revealed that the conformational change of the C‐terminal α‐helix of Rv1546 might contribute to the unique dimer structure. Site‐directed mutagenesis followed by in vitro ribonuclease activity assays was performed to identify catalytic sites of the protein. This experiment suggested that surface residues R63, K84, K88, and R113 are important in the ribonuclease function of Rv1546. In summary, this study presents the structural and functional characterization of Rv1546 and supplies new perspectives for exploiting Rv1546 as a novel drug target for TB treatment. PDB Code(s): 8H0H and 8H2D; [ABSTRACT FROM AUTHOR]

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.

By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus, the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd, encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing. [ABSTRACT FROM AUTHOR]

Streptomyces are soil dwelling bacteria that are notable for their ability to sporulate and to produce antibiotics and other secondary metabolites. Antibiotic biosynthesis is controlled by a variety of complex regulatory networks, involving activators, repressors, signaling molecules and other regulatory elements. One group of enzymes that affects antibiotic synthesis in Streptomyces is the ribonucleases. In this review, the function of five ribonucleases, RNase E, RNase J, polynucleotide phosphorylase, RNase III and oligoribonuclease, and their impact on antibiotic production will be discussed. Mechanisms for the effects of RNase action on antibiotic synthesis are proposed. [ABSTRACT FROM AUTHOR]

Bacteria and archaea respond and adapt to environmental stress conditions by modulating the toxin–antitoxin (TA) system for survival. Within the bacterium Helicobacter pylori, the protein HP0894 is a key player in the HP0894-HP0895 TA system, in which HP0894 serves as a toxin and HP0895 as an antitoxin. HP0894 has intrinsic ribonuclease (RNase) activity that regulates gene expression and translation, significantly influencing bacterial physiology and survival. This activity is influenced by the presence of metal ions such as Mg2+. In this study, we explore the metal-dependent RNase activity of HP0894. Surprisingly, all tested metal ions lead to a reduction in RNase activity, with zinc ions (Zn2+) causing the most significant decrease. The secondary structure of HP0894 remained largely unaffected by Zn2+ binding, whereas structural rigidity was notably increased, as revealed using CD analysis. NMR characterized the Zn2+ binding, implicating numerous His, Asp, and Glu residues in HP0894. In summary, these results suggest that metal ions play a regulatory role in the RNase activity of HP0894, contributing to maintaining the toxin molecule in an inactive state under normal conditions.