Studies on Escherichia coli RNase P RNA with Zn(2+) as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn(2+) as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn(2+) ions in catalysis. Whereas Zn(2+) destabilizes substrate ground state binding to an extent that precludes reliable K(d) determination, [Formula: see text] and Sr(2+) in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn(2+) and [Formula: see text] substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr(2+) on the ribozyme reaction with Zn(2+) as cofactor could be rationalized by a model involving two Sr(2+) ions (or two classes of Sr(2+) ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn(2+). A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr(2+). Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg(2+) entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance

Antisense-Inhibition der bakteriellen RNase P

Ziel der vorliegenden Arbeit war die in vitro- und in vivo-Inhibition bakterieller RNase P mit Antisense-Oligonukleotiden (AS-ON). RNase P ist ein essentielles Ribonukleoproteinenzym, das in allen drei Reichen des Lebens für die Reifung der ptRNAs zuständig ist. Für die Inhibitionsversuche wurde je eine RNase P RNA des Strukturtyps A und B ausgewählt (Typ A: Escherichia coli RNase P RNA; Typ B: Mycoplasma hyopneumoniae RNase P RNA). Bei beiden Strukturtypen bindet die ptRNA an die sogenannte CCA-Bindungsstelle in der P15-Schleife der RNA-Untereinheit als Voraussetzung für eine effiziente Abspaltung der 5´-Flanke der ptRNA. Die CCA-Bindungsstelle ist für die ptRNA-Reifung also von zentraler Bedeutung und sie ist gut zugänglich, da sie an der Oberfläche des Holoenzyms liegt. Die Sequenzen in der Nähe der CCA-Bindungsstelle sind bei verschiedenen Bakterienstämmen nicht hoch konserviert, was die Möglichkeit bietet, AS-ON spezifisch für einen Bakterienstamm oder eine Subgruppe von Bakterien zu konzipieren. Außerdem ist in eukaryontischen P RNAs keine CCA-Bindungsstelle bekannt. Dies alles macht die CCA-Bindungsstelle zu einem interessanten target für Antisense-Strategien zur Bekämpfung pathogener Bakterien. Ausgangspunkt waren haarnadelförmige RNA-AS-ON die teilweise komplementär zur CCA-Bindungsregion der E. coli RNase P RNA und in Anlehnung an ein in E. coli natürlich vorkommendes Antisense-Prinzip entworfen worden waren. Der Versuch, die Strategie der haarnadelförmigen AS-ON auf die Typ B-RNase P RNA von Mycoplasma hyopneumoniae zu übertragen, schlug fehl. Untersuchungen zum Wirkmechanismus der AS-ON auf die E. coli-RNase P RNA zeigten, dass ein einzelsträngiges RNA-AS-ON (RNA-15mer), das nur aus dem zur CCA-Bindungsstelle komplementären Teil des haarnadelförmigen Inhibitors bestand, ein deutlich höheres Inhibitionspotential aufwies. Im weiteren Verlauf wurde das RNA-15mer als Ausgangspunkt für Optimierungen der E. coli-spezifischen Inhibitoren gewählt. Veränderungen in der Länge der Oligonukleotide führten zu dem effektivsten Inhibitor (RNA-14mer, Ki-Wert = 2,2 nM). Da die AS-ON langfristig in vivo eingesetzt werden sollten, wurde im zweiten Teil der Arbeit das Inhibitionspotential Nuklease-stabilisierter Analoga des RNA-14mers untersucht. Für das LNA-14mer (Locked Nucleic Acid) und das RNA 14mer wurde ein vergleichbarer Ki-Wert gefunden, gefolgt von der PNA-Variante (Peptide Nucleic Acid) und DNA-Variante. Um die Spezifität der 14mere zu adressieren, wurden sie an anderen bakteriellen RNase P RNAs getestet, die sich in ihrer Sequenz im Bereich der P15-Region geringfügig von der E. coli-RNase P RNA unterscheiden. Dabei stellte sich heraus, dass die PNA-Variante spezifischer als das RNA-14mer und beide deutlich spezifischer als das LNA-14mer waren. Eine Untersuchung des Assoziationsverhaltens der vier sequenzgleichen 14mere ergab die höchste Assoziationsrate für das PNA-Analogon, gefolgt von der LNA-, RNA- und DNA-Variante. In Bleispaltungsversuchen konnte der Nachweis geführt werden, dass die Inhibition tatsächlich über einen Antisense-Mechanismus abläuft und So wurde für alle vier 14mere die Invasion in die E. coli-P RNA gezeigt, bei der die Helix P15 aufgebrochen wird und es zur Bildung einer Hybridhelix über die gesamte Länge des 14mer-Inhibitors kommt. Aufgrund seiner hohen Assoziationsrate, guten Affinität und Spezifität wurde das PNA-Oligomer für die in vivo-Versuche ausgewählt. PNA-Oligomere können auf Grund der gleichen Kopplungschemie unproblematisch mit invasiven Peptiden verknüpft werden, die die Durchlässigkeit der bakteriellen Zellhüllen für AS-ON verbessern. Im dritten Teil der Arbeit wurde die PNA-Variante an zwei verschiedenen E. coli-Stämmen auf ihr in vivo-Inhibitionspotential untersucht. Das PNA-14mer wurde mit zwei verschiedenen linker-Varianten (dem neuen Glycin und dem Standard AEEA-linker), an das invasive Peptid KFFKFFKFFK gekoppelt. Tests in dem in vitro Standardassay legten nahe, dass weder einer der linker noch das Peptid einen signifikant störenden Einfluss auf die Inhibitionswirkung haben. In in vivo-Experimenten mit dem E. coli-Wildtyp K12 in 10% LB-Medium konnte bei einer Konzentration von 10 µM des PNA-Peptid-Konjugats mit dem Glycin-linker keine Koloniebildung mehr nachgewiesen werden, während das AEEA-linker-Konjugat das Wachstum nur partiell hemmte. Die Inhibitoren zeigten eine qualitativ sehr ähnliche, jedoch stärker ausgeprägte Hemmwirkung auf den LPS-defizienten Stamm AS19. Die unterschiedliche Wirksamkeit veranschaulichte die Bedeutung der äußeren Zellmembran als Hindernis für die zelluläre Aufnahme der PNA-Peptid-Konjugate. Die Ergebnisse zeigen, dass über die Kopplung solcher Peptide die Aufnahme von AS-ON erheblich verbessert werden kann. Die Aufnahme in die Zelle scheint sehr effizient zu erfolgen, da bereits nach 10 min Inkubationszeit in Anwesenheit einer 10 µmolaren Konzentration des PNA-G-Peptids fast keine Koloniebildung mehr beobachtet wurde

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites

Investigation of the catalytic mechanism of RNase P: the role of divalent metal ions and functional groups important for catalysis

The ribonucleoprotein enzyme ribonuclease (RNase) P is an endonuclease that generates the mature 5' -ends of tRNA. Bacterial RNase P is composed of a large RNA subunit (P RNA) and a small protein (P protein). Studies with P RNA from E. coli and B. subtilis have implied a specific role for two or more metal ions in substrate binding and cleavage chemistry. In this study it is demonstrated, for the first time, catalysis by E. coli P RNA with zinc as the sole divalent metal ion cofactor. Although proficient in catalysis, zinc destabilises E•S complex formation. In contrast, strontium inhibits catalysis, but promotes high substrate affinity. Stimulating and inhibitory effects of strontium could be rationalised by a model involving two strontium ions (or two classes), both improving substrate affinity in a cooperative manner, but one of the two inhibiting substrate conversion in a noncompetitive mode with respect to substrate. Further analyses suggest that the inhibition mode of strontium is noncompetitive with respect to zinc. The 2'-OH group at the scissile phosphodiester (nt –1 of ptRNA) contributes to positioning of a catalytic metal ion and may donate a H-bond to the 3'-O leaving group. NMR analyses have indicated that the ribose at nt –1 predominantly populates the C2'-endo conformation. Since the energy barrier for interconversion of C2'- and C3'-endo puckering is expected to be low, it is unknown which conformation is adopted during P RNA catalysis. To address this issue, we analysed cleavage of a ptRNA carrying an locked nucleic acid (LNA) substitution at nt –1, LNA being the only well known substituent that locks the ribose in a C3'-endo puckering. A 2'-methoxy substitution at this position was analysed in parallel, since it is chemically closely related to LNA. Other variants with 2'-fluoro or 2'-deoxy substitution at nt –1 were included in this study. An LNA substitution at nt –1 dramatically reduced (more that a 2'-deoxy) cleavage at the canonical site (-1/+1), while the effects on ground state binding were marginal. A 2'-methoxy substitution completely abolished cleavage at the canonical site. Also, both substituents suppressed cleavage at the site -1/-2. Instead, aberrant cleavage at the site +1/+2 was observed. Since the cleavage at the canonical site of the substrate with LNA at nt –1 had a higher magnesium requirement compared to cleavage at the +1/+2 site, it is likely that the methylene group of LNA inhibit cleavage at the canonical site by sterical interference with a catalytic magnesium ion. The fact that LNA at nt –1 still permitted residual cleavage at the canonical site indicates that the transition state can be reached in the presence of a locked C3'-endo conformation at nt –1. We further tested LNA at nt +1. Here, LNA had only a marginal effect on cleavage chemistry, but significantly reduced ptRNA binding affinity. The binding defect could be overcome at high metal ion concentrations. Again, comparison with a 2'-methoxy and 2'-deoxy modification indicated sterical hindrance by the methylen or methyl group. Hill analysis of metal ion dependence of ptRNA binding revealed a higher metal ion cooperativity for the LNA variant compared to the unmodified one, indicating that the methylene group sterically interferes, directly or indirectly, with metal ion binding to at least one site crucial for E•S complex formation. Functional groups within the P RNA and/or ptRNA are essential for substrate binding and cleavage and they can be mapped by modification interference studies: nucleotide analogue interference mapping (NAIM) and suppression (NAIS). For this purpose, an RNA chimera consisting of E. coli P RNA and the tRNA 5'-half was constructed. A functional substrate was reconstituted by annealing the tRNA 5'-half with its 3'-half resulting in a cis-cleaving RNA complex. A partially modified RNA pool (RNA chimera) was then synthesised by in vitro transcription. After separation of functional (cis-cleaving) and non-functional molecules, positions within the 3' -half of the P RNA where modifications interfered with processing (cis-cleavage reaction) could be identified. These positions are overlapping to some extent with those found in a previous study, where interference with E. coli P RNA - tRNA binding was analysed. These results are to be expected because functional groups in P RNA important for substrate binding are likewise important for substrate turnover, since binding is a prerequisite for cleavage. A strong interference effect at G 350 was detected only with the cis – cleavage assay. G 350 may represent a position essential for the catalytic step, as evidence was provided that it contributes to the binding of catalytically important magnesium near the active site of P RNA

Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus

© 2015 Elsevier B.V. and Socie;acuteacute& Franc de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Bacterial 6S RNAs competitively inhibit binding of RNA polymerase (RNAP) holoenzymes to DNA promoters, thereby globally regulating transcription. RNAP uses 6S RNA itself as a template to synthesize short transcripts, termed pRNAs (product RNAs). Longer pRNAs (approx. ≥ 10 nt) rearrange the 6S RNA structure and thereby disrupt the 6S RNA:RNAP complex, which enables the enzyme to resume transcription at DNA promoters. We studied 6S RNA of the hyperthermophilic bacterium Aquifex aeolicus, representing the thermodynamically most stable 6S RNA known so far. Applying structure probing and NMR, we show that the RNA adopts the canonical rod-shaped 6S RNA architecture with little structure formation in the central bulge (CB) even at moderate temperatures (≤37°C). 6S RNA:pRNA complex formation triggers an internal structure rearrangement of 6S RNA, i.e. formation of a so-called central bulge collapse (CBC) helix. The persistence of several characteristic NMR imino proton resonances upon pRNA annealing demonstrates that defined helical segments on both sides of the CB are retained in the pRNA-bound state, thus representing a basic framework of the RNA's architecture. RNA-seq analyses revealed pRNA synthesis from 6S RNA in A. aeolicus, identifying 9 to ∼17-mers as the major length species. A. aeolicus 6S RNA can also serve as a template for in vitro pRNA synthesis by RNAP from the mesophile Bacillus subtilis. Binding of a synthetic pRNA to A. aeolicus 6S RNA blocks formation of 6S RNA:RNAP complexes. Our findings indicate that A. aeolicus 6S RNA function in its hyperthermophilic host is mechanistically identical to that of other bacterial 6S RNAs. The use of artificial pRNA variants, designed to disrupt helix P2 from the 3;prime&-CB instead of the 5;prime&-CB but preventing formation of the CBC helix, indicated that the mechanism of pRNA-induced RNAP release has been evolutionarily optimized for transcriptional pRNA initiation in the 5;prime&-CB.DFG (SPP 1258 and IRTG 1384) to RKH, by the DFG (SFB 902 A2) to EDF and the Spanish Ministry of Economy and Competitiveness (BFU2011-23645) to M.S.Peer Reviewe

Insights into 6S RNA in lactic acid bacteria (LAB)

Background: 6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB. Results: We provide a genomic annotation of 6S RNA throughout the Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs. Conclusions: Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.Fil: Cataldo, Pablo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Klemm, Paul. Universitat Phillips; AlemaniaFil: Thüring, Marietta. Universitat Phillips; AlemaniaFil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Hartmann, Roland K.. Universitat Phillips; AlemaniaFil: Lechner, Marcus. Universitat Phillips; Alemani

Genomewide comparison and novel ncRNAs of Aquificales

Background  The Aquificales are a diverse group of thermophilic bacteria that thrive in terrestrial and marine hydrothermal environments. They can be divided into the families Aquificaceae, Desulfurobacteriaceae and Hydrogenothermaceae. Although eleven fully sequenced and assembled genomes are available, only little is known about this taxonomic order in terms of RNA metabolism.  Results  In this work, we compare the available genomes, extend their protein annotation, identify regulatory sequences, annotate non-coding RNAs (ncRNAs) of known function, predict novel ncRNA candidates, show idiosyncrasies of the genetic decoding machinery, present two different types of transfer-messenger RNAs and variations of the CRISPR systems. Furthermore, we performed a phylogenetic analysis of the Aquificales based on entire genome sequences, and extended this by a classification among all bacteria using 16S rRNA sequences and a set of orthologous proteins.  Combining severalin silicofeatures (e.g. conserved and stable secondary structures, GC-content, comparison based on multiple genome alignments) with an in vivo dRNA-seq transcriptome analysis of Aquifex aeolicus, we predict roughly 100 novel ncRNA candidates in this bacterium.  Conclusions  We have here re-analyzed the Aquificales, a group of bacteria thriving in extreme environments, sharing the feature of a small, compact genome with a reduced number of protein and ncRNA genes. We present several classical ncRNAs and riboswitch candidates. By combining in silico analysis with dRNA-seq data of A. aeolicus we predict nearly 100 novel ncRNA candidates

Quantum Annealing and Analog Quantum Computation

We review here the recent success in quantum annealing, i.e., optimization of the cost or energy functions of complex systems utilizing quantum fluctuations. The concept is introduced in successive steps through the studies of mapping of such computationally hard problems to the classical spin glass problems. The quantum spin glass problems arise with the introduction of quantum fluctuations, and the annealing behavior of the systems as these fluctuations are reduced slowly to zero. This provides a general framework for realizing analog quantum computation.Comment: 22 pages, 7 figs (color online); new References Added. Reviews of Modern Physics (in press

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly