ABSTRACT
Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996-4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8-51.5%) in comparison to offline approaches (3.7-50.4%). The sequence coverage was increased to 65.6-85.6 and 69.7-85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.
Subject(s)
RNA, Messenger , RNA, Messenger/analysis , RNA, Messenger/genetics , Nucleotide Mapping/methods , Mass Spectrometry , Chromatography, Liquid , Uridine/analogs & derivatives , Uridine/chemistry , Humans , Ribonuclease T1/metabolismABSTRACT
In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.
Subject(s)
Ascomycota , Ascomycota/metabolism , Edible Grain/genetics , Edible Grain/metabolism , Ribonuclease T1/genetics , Ribonuclease T1/metabolism , Polymorphism, Genetic , Plant Diseases/microbiology , Plant Proteins/metabolismABSTRACT
Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.
Subject(s)
Betaine , Muramidase , Methylamines/chemistry , Muramidase/metabolism , Protein Stability , Ribonuclease T1/metabolism , Solutions , Thermodynamics , Water/chemistryABSTRACT
With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.
Subject(s)
Endoribonucleases/chemistry , RNA, Messenger/chemistry , Sequence Analysis, RNA/methods , Tandem Mass Spectrometry , Chromatography, Liquid/methods , DNA, Complementary , Humans , Oligonucleotides/analysis , RNA, Messenger/genetics , Ribonuclease T1/metabolism , Tandem Mass Spectrometry/methodsABSTRACT
In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.
Subject(s)
Nuclease Protection Assays/methods , RNA Probes , RNA/analysis , Ribonucleases , Nucleic Acid Hybridization , RNA/chemistry , RNA/metabolism , RNA, Messenger/analysis , RNA, Messenger/chemistry , Ribonuclease T1/metabolism , Ribonuclease, Pancreatic/metabolismABSTRACT
RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.
Subject(s)
Mass Spectrometry , Oligonucleotides/analysis , RNA Processing, Post-Transcriptional , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine Deaminase/metabolism , Chromatography, Gel , HEK293 Cells , HeLa Cells , Humans , Mixed Function Oxygenases/metabolism , Oligonucleotides/isolation & purification , RNA, Transfer/biosynthesis , RNA, Transfer/isolation & purification , RNA, Transfer, Val/chemistry , RNA, Transfer, Val/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease T1/metabolismABSTRACT
While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).
Subject(s)
Oligonucleotides/analysis , RNA, Transfer/analysis , Sequence Analysis, RNA/methods , Chromatography, High Pressure Liquid , Gene Library , Ribonuclease T1/metabolism , Software , Tandem Mass SpectrometryABSTRACT
Characterization of mRNA sequences is a critical aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can positively identify a single correct sequence from hundreds of similarly sized sequences. In addition, the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.
Subject(s)
Chromatography, Liquid/methods , Erythropoietin/metabolism , Oligonucleotides/analysis , Polymorphism, Single Nucleotide , RNA, Messenger/metabolism , Tandem Mass Spectrometry/methods , alpha Catenin/metabolism , Colicins/metabolism , DNA-Binding Proteins/metabolism , Endoribonucleases/metabolism , Erythropoietin/genetics , Escherichia coli Proteins/metabolism , High-Throughput Nucleotide Sequencing , Humans , RNA, Messenger/genetics , Ribonuclease T1/metabolism , Sequence Analysis, RNA , Software , alpha Catenin/geneticsABSTRACT
Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.
Subject(s)
Chromatography, Liquid/statistics & numerical data , Oligoribonucleotides/analysis , RNA Processing, Post-Transcriptional , RNA, Transfer, Phe/analysis , Software , Tandem Mass Spectrometry/statistics & numerical data , Alkaline Phosphatase/metabolism , Data Interpretation, Statistical , Oligoribonucleotides/chemistry , Oligoribonucleotides/metabolism , RNA, Transfer, Phe/chemistry , RNA, Transfer, Phe/metabolism , Ribonuclease T1/metabolism , Saccharomyces cerevisiae , Sequence Analysis, RNA/statistics & numerical dataABSTRACT
Preferential solvation is a fundamental parameter for the interpretation of solubility and solute structural stability. The molecular basis for solute-solvent interactions can be obtained through distribution functions, and the thermodynamic connection to experimental data depends on the computation of distribution integrals, specifically Kirkwood-Buff integrals for the determination of preferential interactions. Standard radial distribution functions, however, are not convenient for the study of the solvation of complex, nonspherical solutes, as proteins. Here we show that minimum-distance distribution functions can be used to compute KB integrals while at the same time providing an insightful view of solute-solvent interactions at the molecular level. We compute preferential solvation parameters for Ribonuclease T1 in aqueous solutions of urea and trimethylamine N-oxide (TMAO) and show that, while macroscopic solvation shows that urea is preferentially bound to the protein surface and TMAO is preferentially excluded, both display specific density augmentations at the protein surface in dilute solutions. Therefore, direct protein-osmolyte interactions can play a role in the stability and activity of the protein even for preferentially hydrated systems. The generality of the distribution function and its natural connection to thermodynamic data suggest that it will be useful in general for the study of solvation in mixtures of structurally complex solutes and solvents.
Subject(s)
Ribonuclease T1/chemistry , Solvents/chemistry , Methylamines/chemistry , Molecular Dynamics Simulation , Ribonuclease T1/metabolism , Thermodynamics , Urea/chemistryABSTRACT
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven to be a powerful analytical tool for the characterization of modified ribonucleic acids (RNAs). The typical approach for analyzing modified nucleosides within RNA sequences by mass spectrometry involves ribonuclease digestion followed by LC-MS/MS analysis and data interpretation. Here we describe a new software tool, RNAModMapper (RAMM), to assist in the interpretation of LC-MS/MS data. RAMM is a stand-alone package that requires user-submitted DNA or RNA sequences to create a local database against which collision-induced dissociation (CID) data of modified oligonucleotides can be compared. RAMM can interpret MS/MS data containing modified nucleosides in two modes: fixed and variable. In addition, RAMM can also utilize interpreted MS/MS data for RNA modification mapping back against the input sequence(s). The applicability of RAMM was first tested using total tRNA isolated from Escherichia coli. It was then applied to map modifications found in 16S and 23S rRNA from Streptomyces griseus.
Subject(s)
RNA/analysis , Software , Tandem Mass Spectrometry , Area Under Curve , Chromatography, High Pressure Liquid , Databases, Factual , Nucleosides/chemistry , RNA/metabolism , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/metabolism , RNA, Ribosomal, 23S/analysis , RNA, Ribosomal, 23S/metabolism , ROC Curve , Ribonuclease T1/metabolism , Streptomyces griseus/geneticsABSTRACT
Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original 18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a 13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15 Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15 Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract á .
Subject(s)
Isotope Labeling/methods , RNA/analysis , RNA/chemistry , Tandem Mass Spectrometry/methods , Calibration , Carbon Isotopes/chemistry , Chromatography, Liquid/methods , Escherichia coli/genetics , Isotope Labeling/standards , Nitrogen Isotopes/chemistry , RNA, Transfer/analysis , RNA, Transfer/chemistry , Ribonuclease T1/chemistry , Ribonuclease T1/metabolismABSTRACT
This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T1 (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S1 (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man10GlcNAc2 and Man11GlcNAc2 were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.
Subject(s)
Aspergillus oryzae/enzymology , Biotechnology , Fermentation , Aspartic Acid Endopeptidases/isolation & purification , Aspartic Acid Endopeptidases/metabolism , Aspergillus oryzae/metabolism , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Glucan 1,4-alpha-Glucosidase/isolation & purification , Glucan 1,4-alpha-Glucosidase/metabolism , Humans , Japan , Ribonuclease T1/isolation & purification , Ribonuclease T1/metabolism , Single-Strand Specific DNA and RNA Endonucleases/isolation & purification , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Trypsinogen/metabolismABSTRACT
The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem-loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem-loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K(+) and Mg(2+) ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.
Subject(s)
G-Quadruplexes , MicroRNAs/chemistry , MicroRNAs/metabolism , Ribonuclease III/metabolism , Base Sequence , Gene Expression Regulation , Humans , MCF-7 Cells , MicroRNAs/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Ribonuclease T1/metabolism , Transcription, GeneticABSTRACT
Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.
Subject(s)
Antineoplastic Agents/pharmacology , Bacteria/drug effects , Bacteria/genetics , Cisplatin/pharmacology , Inverted Repeat Sequences , RNA, Bacterial/genetics , RNA, Ribosomal/genetics , Bacteria/metabolism , Escherichia coli/genetics , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribonuclease T1/metabolismABSTRACT
Toxins have been thoroughly studied for their use as therapeutic agents in search of an improvement in toxic efficiency together with a minimization of their undesired side effects. Different studies have shown how toxins can follow different intracellular pathways which are connected with their cytotoxic action inside the cells. The work herein presented describes the different pathways followed by the ribotoxin α-sarcin and the fungal RNase T1, as toxic domains of immunoconjugates with identical binding domain, the single chain variable fragment of a monoclonal antibody raised against the glycoprotein A33. According to the results obtained both immunoconjugates enter the cells via early endosomes and, while α-sarcin can translocate directly into the cytosol to exert its deathly action, RNase T1 follows a pathway that involves lysosomes and the Golgi apparatus. These facts contribute to explaining the different cytotoxicity observed against their targeted cells, and reveal how the innate properties of the toxic domain, apart from its catalytic features, can be a key factor to be considered for immunotoxin optimization.
Subject(s)
Endoribonucleases/metabolism , Fungal Proteins/metabolism , Immunoconjugates/metabolism , Ribonuclease T1/metabolism , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Circular Dichroism , Colonic Neoplasms/metabolism , Endoribonucleases/genetics , Fungal Proteins/genetics , Humans , Immunoconjugates/genetics , Immunotoxins/genetics , Immunotoxins/metabolism , Microscopy, Fluorescence , Protein Transport , Ribonuclease T1/geneticsABSTRACT
Extracellular low-molecular weight guanyl-preferring ribonucleases (LMW RNases) of Bacillus sp. comprise a group of hydrolytic enzymes that share highly similar structural and catalytic characteristics with barnase, a ribonuclease from Bacillus amyloliquefaciens, and binase, a ribonuclease from Bacillus intermedius. Although the physical-chemical and catalytic properties of Bacillus guanyl-preferring ribonucleases are very similar, there is considerably more variation in the environmental conditions that lead to the induction of the genes encoding these RNases. Based on structural differences of their genes the guanyl-preferring ribonucleases have been sub-divided into binase-like and barnase-like groups. Here we show the ability of the key regulator of phosphate deficiency response, PhoP, to direct the transcription of the binase-like RNases but not barnase-like RNases. These results, together with our demonstration that binase-like RNases are induced in response to phosphate starvation, allow us to categorise this group of ribonucleases as new members of Bacillus PhoP regulon. In contrast, the barnase-like ribonucleases are relatively insensitive to the phosphate concentration and the environmental conditions that are responsible for their induction, and the regulatory elements involved, are currently unknown.
Subject(s)
Bacillus/genetics , Regulon/genetics , Ribonuclease T1/genetics , Amino Acid Sequence , Bacillus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Nucleotide Motifs , Phylogeny , Position-Specific Scoring Matrices , Promoter Regions, Genetic , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonuclease T1/chemistry , Ribonuclease T1/classification , Ribonuclease T1/metabolism , Sequence AlignmentABSTRACT
RNA has different levels of structural organization. The primary structure is the linear order of the nucleotide monomers, the RNA sequence. During transcription process, the partially synthesized RNA is folded by base-pairing and thermodynamic intramolecular or intermolecular interactions. This results in a dynamic spreading of a secondary structure along the length of the transcribed section of the RNA. The analysis of both primary or secondary structures requires the RNA end-labeling either at its 5' end using a kinase reaction with [gamma-32P]ATP, or at its 3' end using an RNA ligation reaction with [32P]pCp. End-labeled RNAs are then gradually breakdown using hydrolysing chemicals or a variety of enzymes targeting specific RNA sequences and secondary structure. The most commonly used enzymes are RNase A, T1, and V1. The partial digestion of the RNA reveals a mix of truncated RNA fragments of different lengths, called RNA ladder. The products are then separates through a high resolution gel system and subjected to autoradiographic analysis. Each visible fragment is labeled at one end, but comprises an enzyme specific sequence at the other end. Final comparison of the detected RNA ladders reveals a hypothetical model of the secondary RNA structure under assay conditions.
Subject(s)
RNA/analysis , RNA/chemistry , Nucleic Acid Conformation , RNA/metabolism , Ribonuclease T1/metabolism , Ribonucleases/metabolismABSTRACT
Recent progress in the synthesis of nucleotides from prebiotically plausible precursors has opened up new ways to explain the origin of genetic matter. Mechanisms for the polymerization of nucleotides without the help of catalysts are, however, rare. Complementary to the experiments done by Costanzo et al., we found that drying 3',5'-cyclic GMP leads to poly-G RNA strands with lengths of up to 40 nucleotides. We also show that the polymerization to long RNA strands is considerably more efficient under dry conditions than for cGMP polymerization in water. The length depends on the incubation time of dry nucleotides at temperatures of 40-80 °C. No enzymes or other catalysts are needed for successful polymerization.
Subject(s)
Cyclic GMP/metabolism , RNA/metabolism , Polymerization , RNA/chemistry , Ribonuclease T1/metabolism , Temperature , Water/chemistryABSTRACT
The length of the CAG-repeat region in the huntingtin mRNA is predictive of Huntington's disease. Structural studies of CAG-repeat-containing RNAs suggest that these sequences form simple hairpin structures; however, in the context of the full-length huntingtin mRNA, CAG repeats may form complex structures that could be targeted for therapeutic intervention. We examined the structures of transcripts spanning the first exon of the huntingtin mRNA with both healthy and disease-prone repeat lengths. In transcripts with 17-70 repeats, the CAG sequences base paired extensively with nucleotides in the 5' UTR and with conserved downstream sequences including a CCG-repeat region. In huntingtin transcripts with healthy numbers of repeats, the previously observed CAG hairpin was either absent or short. In contrast, in transcripts with disease-associated numbers of repeats, a CAG hairpin was present and extended from a three-helix junction. Our findings demonstrate the profound importance of sequence context in RNA folding and identify specific structural differences between healthy and disease-inducing huntingtin alleles that may be targets for therapeutic intervention.