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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.

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).

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.

RNA:DNA hybrids are important intermediates in cellular processes. Smolka et al. establish controls to test the specificity of the S9.6 anti-RNA:DNA hybrid antibody in imaging approaches. They found that S9.6 immunofluorescence signal derives primarily from ribosomal RNA, not RNA:DNA hybrids., The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin &alpha, sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide-bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG-mono-sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide-bridging conjugation to give the PEGylated proteins. Interferon-β1b (IFN-β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging-conjugation during refolding. The PEG-IFN-β1b was isolated by ion-exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN-β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN-β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.

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.

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, specically Kirkwood-Bu integrals for the determination of preferential interaction or exclusion. Standard radial distribution function functions, however, are not convenient for the study of the solvation of complex, non-spherical solutes, as proteins structures. Here we show that minimum-distance distribution functions can be used to compute KB integrals while at the same time providing a rich 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 specic density augmentations at the protein surface. 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 suggests that it will be useful in general for the study of solvation in mixtures of structurally complex solutes and solvents.

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.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.

A group of enzymes, mostly hydrolases or certain transferases, utilize one or a few side-chain carboxyl groups of Asp and/or Glu as part of the catalytic machinery at their active sites. This review follows mainly the trail of studies performed by the author and his colleagues on the structure and function of such enzymes, starting from ribonuclease T1, then extending to three major types of carboxyl peptidases including aspartic peptidases, glutamic peptidases and serine-carboxyl peptidases.

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.

Slow protein folding processes during which kinetic folding intermediates occur for an extended time can lead to aggregation and dysfunction in living cells. Therefore, protein folding helpers have evolved, which prevent proteins from aggregation and/or speed up folding processes. In this study, we present the structural characterization of a long-living transient folding intermediate of RNase T1 (S54G/P55N) by time-resolved NMR spectroscopy. NMR resonances of this kinetic folding intermediate could be assigned mainly by a real-time 3D BEST-HNCA. These assignments were the basis to investigate the interaction sites between the protein folding helper enzyme SlyD(1-165) (SlyD*) from Escherichia coli (E. coli) and this kinetic intermediate at a residue resolution. Thus, we investigated the Michaelis-Menten complex of this enzyme reaction, because the NMR data acquisition was performed during the actual catalysis. The interaction surface of the transient folding intermediate is restricted to a region around the peptidyl-prolyl bond (Y38-P39), whose isomerization is catalyzed by SlyD*. The interaction surface regarding SlyD* extends from specific amino acids of the FKBP domain forming the peptidyl-prolyl cis/trans-isomerase active site to almost the entire IF domain. This illustrates an effective interplay between the two functional domains of SlyD* to facilitate protein folding catalysis. published

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions.

Supramolecular assembly is a powerful strategy used by nature to build nanoscale architectures with predefined sizes and shapes. With synthetic systems, however, numerous challenges remain to be solved before precise control over the synthesis, folding and assembly of rationally designed three-dimensional nano-objects made of RNA can be achieved. Here, using the transfer RNA molecule as a structural building block, we report the design, efficient synthesis and structural characterization of stable, modular three-dimensional particles adopting the polyhedral geometry of a non-uniform square antiprism. The spatial control within the final architecture allows the precise positioning and encapsulation of proteins. This work demonstrates that a remarkable degree of structural control can be achieved with RNA structural motifs for the construction of thermostable three-dimensional nano-architectures that do not rely on helix bundles or tensegrity. RNA three-dimensional particles could potentially be used as carriers or scaffolds in nanomedicine and synthetic biology. Whereas synthetic DNA nanostructures are widely studied, the use of RNA as a structural building block is much less common. Now, it has been shown that tRNA molecules can be designed to assemble into a rigid and thermally stable square antiprism structure that may prove useful for delivery applications inside cells.

Because of their central importance for understanding enzymatic mechanisms, pK(a) values are of great interest in biochemical research. It is common practice to determine pK(a) values of amino acid residues in proteins from NMR or FTIR titration curves by determining the pH at which the protonation probability is 50%. The pH dependence of the free energy required to protonate this residue is then determined from the linear relationship DeltaG(prot) = RT ln 10 (pH-pK(a)), where R is the gas constant and T the absolute temperature. However, this approach neglects that there can be important electrostatic interactions in the proteins that can shift the protonation energy. Even if the titration curves seem to have a standard sigmoidal shape, the protonation energy of an individual site in a protein may depend nonlinearly on pH. To account for this nonlinear dependence, we show that it is required to introduce pK(a) values for individual sites in proteins that depend on pH. Two different definitions are discussed. One definition is based on a rearranged Henderson-Hasselbalch equation, and the other definition is based on an equation that was used by Tanford and Roxby to approximate titration curves of proteins. In the limiting case of weak interactions, the two definitions lead to nearly the same pK(a) value. We discuss how these two differently defined pK(a) values are related to the free energy change required to protonate a site. Using individual site pK(a) values, we demonstrate on simple model systems that the interactions between protonatable residues in proteins can help to maintain the energy required to protonate a site in the protein nearly constant over a wide pH range. We show with the example of RNase T1 that such a mechanism to keep the protonation energy constant is used in enzymes. The pH dependence of pK(a) values may be an important concept in enzyme catalysis. Neglecting this concept, important features of enzymes may be missed, and the enzymatic mechanism may not be fully understood.

Supplementari Data http://nar.oxfordjournals.org/content/suppl/2009/10/12/gkp553.DC1, It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5′ untranslated region could ultimately represent a new mechanism of action of micro-RNAs., Ministerio de Ciencia e Innovación BIO2007-60106, BIO2004-06114 Proyecto Excelencia Comunidad Autónoma de Andalucía CVI-03050; FISS (CIBERehd); FIPSE 36549/06 for work in Granada; National Institutes of Health grant NIH/NIDA K01 DAO18262 for work in Doylestown, Pennsylvania. Funding for open access charge: BIO-2007, 60106 (IPB-LN) CSIC. MCyT.

Recent studies have identified RNA transcripts arising from mammalian telomeres with the transcript of the C-rich strand (r(5′-UUAGGG-3′)n) being much more abundant than transcripts of the G-rich strand. Here we used transmission electron microscopy, CD, and nuclease digestion to investigate the structure of ∼640-nucleotide (nt) RNA transcripts of the C-rich and G-rich strands of mammalian telomeric DNA. The CD spectrum of the C-rich RNA in low salt (10 mm KCl) or high salt (100 mm KCl) was typical of mixed sequence RNA, whereas the CD spectrum for the G-rich RNA differed with changes characteristic of parallel G-quadruplexes at the higher salt concentration. Electron microscopy visualization of the C-rich RNA revealed relatively extended unstructured molecules 59.7 ± 17.8 nm in length and with a width consistent with single-stranded RNA following metal coating. In contrast, the G-rich RNA was observed as round particles and short, thick rods with the rods being most prevalent in high salt conditions and absent in low salt. The rods were 22.7 ± 4.8 nm in length and 7.6 nm in width. Digestion of the G-rich RNA with T1 RNA nuclease revealed a ladder of bands whose sizes were integral multiples of 24 nt plus a 4-nt overhang. These observations suggest a model in which G-rich telomeric RNA folds into chains of particles each consisting of four (UUAGGG) repeats stabilized by parallel G-quartets and joined by UUA linkers. These chains further condense to form short rods and round particles.

The T7RNA polymerase (RNAP) elongation complex (EC) pauses and is destabilized at a unique 8 nucleotide (nt) sequence found at the junction of the head-to-tail concatemers of T7 genomic DNA generated during T7 DNA replication. The paused EC may recruit the T7 DNA processing machinery, which cleaves the concatemerized DNA within this 8 nt concatemer junction (CJ). Pausing of the EC at the CJ involves structural changes in both the RNAP and transcription bubble. However, these structural changes have not been fully defined, nor is it understood how the CJ sequence itself causes the EC to change its structure, to pause, and to become less stable. Here we use solution and RNAP-tethered chemical nucleases to probe the CJ transcript and changes in the EC structure as the polymerase pauses and terminates at the CJ. Together with extensive mutational scanning of regions of the polymerase that are likely to be involved in recognition of the CJ, we are able to develop a description of the events that occur as the EC transcribes through the CJ and subsequently pauses. In this process, a local change in the structure of the transcription bubble drives a large change in the architecture of the EC. This altered EC structure may then serve as the signal that recruits the processing machinery to the CJ.

RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30 – 200 s via non-native intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.

R-loops have been described in vivo at the immunoglobulin class switch sequences and at prokaryotic and mitochondrial origins of replication. However, the biochemical mechanism and determinants of R-loop formation are unclear. We find that R-loop formation is nearly eliminated when RNase T1 is added during transcription but not when it is added afterward. Hence, rather than forming simply as an extension of the RNA-DNA hybrid of normal transcription, the RNA must exit the RNA polymerase and compete with the nontemplate DNA strand for an R-loop to form. R-loops persist even when transcription is done in Li+ or Cs+, which do not support G-quartet formation. Hence, R-loop formation does not rely on G-quartet formation. R-loop formation efficiency decreases as the number of switch repeats is decreased, although a very low level of R-loop formation occurs at even one 49-bp switch repeat. R-loop formation decreases sharply as G clustering is reduced, even when G density is kept constant. The critical level for R-loop formation is approximately the same point to which evolution drove the G clustering and G density on the nontemplate strand of mammalian switch regions. This provides an independent basis for concluding that the primary function of G clustering, in the context of high G density, is R-loop formation.

Recently, artificial ribonucleases (aRNases)--conjugates of oligodeoxyribonucleotides and peptide (LR)(4)-G-amide--were designed and assessed in terms of the activity and specificity of RNA cleavage. The conjugates were shown to cleave RNA at Pyr-A and G-X sequences. Variations of oligonucleotide length and sequence, peptide and linker structure led to the development of conjugates exhibiting G-X cleavage specificity only. The most efficient catalyst is built of nonadeoxyribonucleotide of unique sequence and peptide (LR)(4)-G-NH(2) connected by the linker of three abasic deoxyribonucleotides (conjugate pep-9). Investigation of the cleavage specificity of conjugate pep-9 showed that the compound is the first single-stranded guanine-specific aRNase, which mimics RNase T1. Rate enhancement of RNA cleavage at G-X linkages catalysed by pep-9 is 10(8) compared to non-catalysed reaction, pep-9 cleaves these linkages only 10(5)-fold less efficiently than RNase T1 (k(cat_RNase T1)/k(cat)_(pep)(-9) = 10(5)).

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.

The separation of biologically active, pure, and specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA identity and/or abundance are difficult. We have discovered that the enzymatic digestion of an individual tRNA by a ribonuclease (e.g., RNase T1) will generate digestion products unique to that particular tRNA, and we show that a comparison of an organism's complete complement of tRNA RNase digestion products yields a set of unique or “signature” digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. Detection is facilitated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and proof-of-principle is demonstrated on the whole tRNA pool from Escherichia coli. This method will enable the individual identification of tRNA isoacceptors without requiring specific affinity purification or extensive chromatographic and/or electrophoretic purification. Further, experimental identifications of tRNAs or other RNAs will now be possible using this signature digestion product approach in a manner similar to peptide mass fingerprinting used in proteomics, allowing RNomic studies of RNA at the post-transcriptional level.

In bacteria, ribosome-bound Trigger Factor assists the folding of newly synthesized proteins. The N-terminal domain (N) of Trigger Factor mediates ribosome binding, whereas the middle domain (P) harbors peptidyl-prolyl isomerase activity. The function of the C-terminal domain (C) has remained enigmatic due to structural instability in isolation. Here, we have characterized a stabilized version of the C domain (C(S)), designed on the basis of the recently solved atomic structure of Trigger Factor. Strikingly, only the isolated C(S) domain or domain combinations thereof (NC(S), PC(S)) revealed substantial chaperone activity in vitro and in vivo. Furthermore, to disrupt the C domain without affecting the overall Trigger Factor structure, we generated a mutant (Delta53) by deletion of the C-terminal 53 amino acid residues. This truncation caused the complete loss of the chaperone activity of Trigger Factor in vitro and severely impaired its function in vivo. Therefore, we conclude that the chaperone activity of Trigger Factor critically depends on its C-terminal domain as the central structural chaperone module. Intriguingly, a structurally similar module is found in the periplasmic chaperone SurA and in MPN555, a protein of unknown function. We speculate that this conserved module can exist solely or in combination with additional domains to fulfill diverse chaperone functions in the cell.

A regulatable promoter has been stably integrated into a human embryonic kidney cell line. The promoter is a pol III mouse promoter and is under the control of ponasterone A, an ecdysone inducer. The promoter controls transcription of an external guide sequence (EGS) targeted against Rpp38, a protein subunit of human RNase P, or of lamin A/C, a gene product located in the nucleus. The amounts of protein of both gene products are severely reduced when the EGSs are made. Several other, but not all, of the protein subunits of RNase P are also inhibited in both mRNA and protein levels when Rpp38 mRNA is targeted.

The bifunctional reagent ethyl 4-azidobenzoylaminoacetimidate was used to crosslink specifically ribosomal protein S1 to 16-S RNA within 30-S subunits. The reagent was attached to isolated protein S1. The modified protein was reassociated with protein-S1-depleted 30-S subunits and then crosslinked to the RNA molecule. The covalently bound 16-S RNA-protein S1 complex was isolated and the RNA fragment C-U-A-A-C-G-C-G-U-U-A-A-G-U-C-G-A-C-C-G-C-C-U-G-G-G-G-A-G (positions 861-889) was characterized to be crosslinked to protein S1.

The nucleotide sequences of wheat embryo 5.8-S and 5-S rRNAs have been determined with the use of several techniques, including classic analysis of oligonucleotides generated by ribo-nuclease T1 and resolution on gels of termiNatly labelled RNA partially degraded with ribonucleases or with chemical reagents. The sequence of wheat embryo 5.8-S rRNA was found to be p(C)-A-C- 5.8-S RNAs, the accommodation of this sequence by current secondary structural models, and the significance of the observed post-transcriptional modification pattern are discussed. The sequence . This sequence is compared to 5-S rRNA Sequences previously published for wheat and several other angiosperms.

Overexpression of the rntA cDNA encoding RNase T1 derived from A. oryzae causes severe growth inhibition in S. cerevisiae. We previously reported that most S. cerevisiae mutant strains defective in translocation into the ER, ER-Golgi transport and vacuole formation exhibited hypersensitivity to expression of RNase T1. Screening for S. cerevisiae mutants that showed RNase T1 hypersensitivity resulted in the isolation of 38 (rns) mutant strains. Some of these mutants showed a variety of phenotypes including temperature-sensitive growth, hypersensitivity to G418, defect in invertase glycosylation and fragmented vacuoles. We identified the genes mutated in three of the rns mutants, rns1, rns2, and rns3, as DSL1, UMP1, and SEC17, respectively. Fluorescence microscopic observation showed that GFP or myc-tagged Rns1p was localized at the nuclear region in the cell. Two-hybrid screening revealed the interaction of Rns1p with a transcription factor Cin5p and a functionally unknown Ylr440cp. It was observed that HA-tagged Ylr440cp was localized to the ER and nuclear envelope.

The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.

The 5′ untranslated leader region of retroviral RNAs contains noncoding information that is essential for viral replication, including signals for transcriptional transactivation, splicing, primer binding for reverse transcription, dimerization of the genomic RNA, and encapsidation of the viral RNA into virions. These RNA motifs have considerable structural and functional overlap. In this study, we investigate the conformational dynamics associated with the use and silencing of a sequence in HIV-2 RNA that is involved in genomic RNA dimerization called stem–loop 1 (SL1) and its relationship with a flanking sequence that is known to be important for encapsidation of viral RNAs. We demonstrate that a long-distance intramolecular interaction between nucleotides located upstream of the primer-binding site domain and nucleotides encompassing the Gag translation start codon functionally silences SL1 as a dimerization element. This silencing can be relieved by mutation or by hybridization of an oligonucleotide that disrupts the long-distance interaction. Furthermore, we identify a palindrome within the packaging/encapsidation signal Ψ (just 5′ of SL1) that can either serve as an efficient dimerization signal itself, or can mediate SL1 silencing through base pairing with SL1. These results provide a tangible link between the functions of genomic RNA dimerization and encapsidation, which are known to be related, but whose physical relationship has been unclear. A model is proposed that accounts for observations of dimerization, packaging, and translation of viral RNAs during different phases of the viral replication cycle.

Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.

The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 microM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are complementary and, on photolysis, incorporate into neighboring ribosomal components. Here we report on photocrosslinking results obtained with PHONTs targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093). Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093 to U1712/1716 and U1926, that are clearly incompatible with published crystal structures of 50S subunits. These results provide strong evidence that within the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion, accessing large areas of the ribosome, and (b) the lateral arm can bend about the noncanonical helix at its center. Such motions may have functional relevance in identifying regions that undergo major conformational change as the ribosome moves through its catalytic cycle.

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5′ exon—intron part, rather than the intron—3′ exon part, of pre-mRNA was observed.

In the Bromoviridae family of plant viruses, trinucleotide hairpin loops play an important role in RNA transcription. Recently, we reported that Brome mosaic virus (BMV) subgenomic (sg) transcription depended on the formation of an unusual triloop hairpin. By native gel electrophoresis, enzymatic structure probing, and NMR spectroscopy it is shown here that in the absence of viral replicase the hexanucleotide loop 5'C1AUAG5A3' of this RNA structure can adopt a pseudo trinucleotide loop conformation by transloop base pairing between C1 and G5. By means of in vitro replication assays using partially purified BMV RNA-dependent RNA polymerase (RdRp) it was found that other base pairs contribute to sg transcription, probably by stabilizing the formation of this pseudo triloop, which is proposed to be the primary element recognized by the viral replicase. The BMV pseudo triloop structure strongly resembles iron-responsive elements (IREs) in cellular messenger RNAs and may represent a general protein-binding motif. In addition, in vitro replication assays showed that the BMV sg hairpin is functionally equivalent to the minus-strand core promoter hairpin stem-loop C at the 3' end of BMV RNAs. Replacement of the sg hairpin by stem-loop C yielded increased sg promoter activity whereas replacement of stem-loop C by the sg hairpin resulted in reduced minus-strand promoter activity. We conclude that AUA triloops represent the common motif in the BMV sg and minus-strand promoters required for recruitment of the viral replicase. Additional sequence elements of the minus-strand promoter are proposed to direct the RdRp to the initiation site at the 3' end of the genomic RNA.