25 results on '"Kaberdin V"'
Search Results
2. The long-term survival of Acinetobacter baumannii ATCC 19606T under nutrient-deprived conditions does not require the entry into the viable but non-culturable state
- Author
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Bravo, Z., Orruño, M., Parada, C., Kaberdin, V. R., Barcina, I., and Arana, I.
- Published
- 2016
- Full Text
- View/download PDF
3. Quaternary structure and biochemical properties of mycobacterial RNase E/G
- Author
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M. E., Zeller, Csanadi, A., Miczak, A., Rose, T., Bizebard, T., Kaberdin, V., and Gauthier, Laurence
- Subjects
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology - Published
- 2007
4. Towards new antituberculotic targets: biochemical characterisation of mycobacterial RNase E/G
- Author
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Csanadi, A., primary, Zeller, M.-E., additional, Miczak, A., additional, Rose, T., additional, Bizebard, T., additional, and Kaberdin, V. R., additional
- Published
- 2008
- Full Text
- View/download PDF
5. Probing the substrate specificity of Escherichia coli RNase E using a novel oligonucleotide-based assay
- Author
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Kaberdin, V. R., primary
- Published
- 2003
- Full Text
- View/download PDF
6. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.
- Author
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Miczak, A, primary, Kaberdin, V R, additional, Wei, C L, additional, and Lin-Chao, S, additional
- Published
- 1996
- Full Text
- View/download PDF
7. Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding.
- Author
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Vytvytska, O, Moll, I, Kaberdin, V R, von Gabain, A, and Bläsi, U
- Abstract
The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.
- Published
- 2000
8. RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme.
- Author
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Kaberdin, V R, Chao, Y H, and Lin-Chao, S
- Abstract
Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.
- Published
- 1996
9. Composition and conservation of the mRNA-degrading machinery in bacteria
- Author
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Kaberdin Vladimir R, Singh Dharam, and Lin-Chao Sue
- Subjects
Medicine - Abstract
Abstract RNA synthesis and decay counteract each other and therefore inversely regulate gene expression in pro- and eukaryotic cells by controlling the steady-state level of individual transcripts. Genetic and biochemical data together with recent in depth annotation of bacterial genomes indicate that many components of the bacterial RNA decay machinery are evolutionarily conserved and that their functional analogues exist in organisms belonging to all kingdoms of life. Here we briefly review biological functions of essential enzymes, their evolutionary conservation and multienzyme complexes that are involved in mRNA decay in Escherichia coli and discuss their conservation in evolutionarily distant bacteria.
- Published
- 2011
- Full Text
- View/download PDF
10. Analysis of Acinetobacter baumannii survival in liquid media and on solid matrices as well as effect of disinfectants.
- Author
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Bravo Z, Orruño M, Navascues T, Ogayar E, Ramos-Vivas J, Kaberdin VR, and Arana I
- Subjects
- Acinetobacter baumannii radiation effects, Culture Media chemistry, Environmental Microbiology, Microbial Viability radiation effects, Temperature, Acinetobacter baumannii drug effects, Acinetobacter baumannii physiology, Disinfectants pharmacology, Microbial Viability drug effects
- Abstract
Background: Acinetobacter baumannii is a cause of healthcare-associated infections and has considerable potential to survive on inanimate hospital surfaces under hostile conditions (e.g. disinfection or desiccation)., Aim: To learn more about its survival strategy and capacity to persist in liquid media and on surfaces mimicking hospital environments., Methods: The effect of temperature, nutrient deprivation, permanence on inanimate surfaces, and exposure to disinfectants on the survival of four A. baumannii strains (ATCC 19606
T and three clinical isolates) was studied by monitoring the number of total and viable cells using fluorescent microscopy and of culturable cells by standard cultures., Findings: Bacterial survival was differentially affected by temperature (cells maintained at 20°C remained culturable at least within 30 days) and physical environment (desiccation favoured cell resistance to stress at 37°C). Moreover, persistence was associated with two adaptation patterns: one linked to entry into the viable but non-culturable state, whereas the other apparently followed a bust-and-boom model. During a study on the effect of disinfectant (commercial bleach and quaternary ammonium compounds), it was found that treatment with these antibacterial compounds did not eliminate A. baumannii populations and provoked the reduction of culturable populations, although a fraction of cells remained culturable., Conclusion: The ability to persist for long periods on different surfaces, mimicking those usually found in hospitals, along with A. baumannii's capacity to survive after a disinfection process may account for the recurrent outbreaks in intensive care units., (Copyright © 2019 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
11. Changes in the Vibrio harveyi Cell Envelope Subproteome During Permanence in Cold Seawater.
- Author
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Parada C, Orruño M, Kaberdin V, Bravo Z, Barcina I, and Arana I
- Subjects
- Adaptation, Physiological, Bacterial Proteins physiology, Cell Membrane physiology, Colony Count, Microbial, Cytochrome b Group, Ferritins, Membrane Proteins physiology, Microbial Viability, Proteome physiology, Time Factors, Vibrio chemistry, Vibrio cytology, Bacterial Proteins chemistry, Cell Membrane chemistry, Cold Temperature, Membrane Proteins chemistry, Proteome chemistry, Seawater microbiology, Vibrio physiology
- Abstract
Previous work demonstrated that physiological, morphological, and gene expression changes as well as the time-dependent entry into the viable but not culturable (VBNC) state are used by Vibrio species to survive and cope with diverse stress conditions including seasonal temperature downshifts and starvation. To learn more about the nature and specific contribution of membrane proteins to cell adaptation and survival, we analyzed variations in the protein composition of cell envelope and related them to morphological and physiological changes that were taking place during the long-term permanence of Vibrio harveyi in seawater microcosm at 4 °C. We found that after 21 days of permanence, nearly all population (ca. 99 %) of V. harveyi acquired the VBNC phenotype. Although the size of V. harveyi cells gradually decreased during the incubation time, we found that this morphological change was not directly related to their entry into the VBNC state. Our proteomic study revealed that the level of membrane proteins playing key roles in cellular transport, maintenance of cell structure, and in bioenergetics processes remained unchanged along starvation at low temperature, thus suggesting that V. harveyi might need these proteins for the long-term survival and/or for the resuscitation process. On a contrary, the level of two proteins, elongation factor Tu (EF-TU) and bacterioferritin, greatly increased reaching the maximal values by the end of the incubation period. We further discuss the above data with respect to the putative roles likely exerted by membrane proteins during transition to and maintaining of the VBNC state.
- Published
- 2016
- Full Text
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12. Translation initiation factor a/eIF2(-gamma) counteracts 5' to 3' mRNA decay in the archaeon Sulfolobus solfataricus.
- Author
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Hasenöhrl D, Lombo T, Kaberdin V, Londei P, and Bläsi U
- Subjects
- Base Sequence, DNA Primers, Protein Binding, RNA, Messenger metabolism, Ribosomes metabolism, Eukaryotic Initiation Factor-2 metabolism, RNA, Messenger genetics, Sulfolobus solfataricus genetics
- Abstract
The trimeric translation initiation factor a/eIF2 of the crenarchaeon Sulfolobus solfataricus is pivotal for binding of initiator tRNA to the ribosome. Here, we present in vitro and in vivo evidence that the a/eIF2 gamma-subunit exhibits an additional function with resemblance to the eukaryotic cap-complex. It binds to the 5'-triphosphate end of mRNA and protects the 5' part from degradation. This unprecedented capacity of the archaeal initiation factor further indicates that 5' --> 3' directional mRNA decay is a pathway common to all domains of life.
- Published
- 2008
- Full Text
- View/download PDF
13. The AGUAAA motif in cspA1/A2 mRNA is important for adaptation of Yersinia enterocolitica to grow at low temperature.
- Author
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Neuhaus K, Anastasov N, Kaberdin V, Francis KP, Miller VL, and Scherer S
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Consensus Sequence, Down-Regulation, Heat-Shock Response, Molecular Sequence Data, RNA, Messenger genetics, RNA, Messenger metabolism, Yersinia enterocolitica genetics, Yersinia enterocolitica metabolism, Yersinia enterocolitica physiology, Adaptation, Physiological, Amino Acid Motifs, Bacterial Proteins chemistry, Cold Temperature, RNA, Messenger chemistry, Yersinia enterocolitica growth & development
- Abstract
Acclimatization of the psychrotolerant Yersinia enterocolitica after a cold shock from 30 degrees C to 10 degrees C causes transcription of the major cold shock protein (CSP) bicistronic gene cspA1/A2 to increase by up to 300-fold. Northern blot analysis of cspA1/A2 using four probes that hybridize specifically to different regions of CSP mRNA revealed the appearance of a number of cspA1/A2 transcripts that are smaller than the original transcript and transiently visible at the end of the acclimation period. Primer extension and RNA protection experiments demonstrated that these smaller mRNAs have 5' ends located in the same core sequence (5'-AGUAAA-3') at five different places within the mRNA, indicating preferential cleavage of the CSP mRNA transcripts. A similar result was obtained for cspB of Escherichia coli, containing two such core sequences. Furthermore, this motif is present in the major CSP genes of a variety of Gram-negative and Gram-positive bacteria. We have therefore termed this sequence cold shock cut box (CSC-box). After inserting a CSC-box into a plasmid-bound lacZ gene in Y. enterocolitica, the mRNA of this construct was cleaved within the CSC-box, and a change in this CSC-box from AGUAAA to AGUCCC dramatically reduced cleavage of the mutated lacZ gene. Mutating all CSC-boxes in Y. enterocolitica of a plasmid bound cspA1/A2 dramatically increases the lag time after a cold shock before re-growth occurs. Based on these results, we suggest that the role of the CSC-box is related to downregulation of cspA mRNA after acclimation to low temperature.
- Published
- 2003
- Full Text
- View/download PDF
14. Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli.
- Author
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Vecerek B, Moll I, Afonyushkin T, Kaberdin V, and Bläsi U
- Subjects
- 5,10-Methylenetetrahydrofolate Reductase (FADH2) genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Carrier Proteins genetics, Cyclophilins genetics, Escherichia coli genetics, Escherichia coli Proteins genetics, Gene Expression Regulation, Bacterial, Host Factor 1 Protein genetics, Molecular Chaperones genetics, Molecular Chaperones metabolism, Molecular Sequence Data, Peptidylprolyl Isomerase, Protein Biosynthesis, Proteomics methods, RNA Stability, RNA, Small Nuclear genetics, RNA, Small Nuclear metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Host Factor 1 Protein metabolism, Iron metabolism, RNA, Messenger metabolism
- Abstract
The Escherichia coli Sm-like host factor I (Hfq) is thought to play direct and indirect roles in post-transcriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq- background. The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.
- Published
- 2003
- Full Text
- View/download PDF
15. Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli.
- Author
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Walsh AP, Tock MR, Mallen MH, Kaberdin VR, von Gabain A, and McDowall KJ
- Subjects
- Base Sequence, Kinetics, Molecular Sequence Data, Nucleic Acid Conformation, Oligonucleotides chemistry, Oligonucleotides metabolism, Phosphorylation, RNA, Messenger chemistry, 3' Untranslated Regions, Endoribonucleases metabolism, Escherichia coli enzymology, Poly A metabolism, RNA, Messenger metabolism
- Abstract
RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.
- Published
- 2001
- Full Text
- View/download PDF
16. Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E.
- Author
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Kaberdin VR, Walsh AP, Jakobsen T, McDowall KJ, and von Gabain A
- Subjects
- Amino Acid Motifs, Base Sequence, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Nucleic Acid Conformation, Protein Structure, Tertiary, RNA Precursors chemistry, RNA, Messenger metabolism, Substrate Specificity, Arginine chemistry, Endoribonucleases chemistry, Escherichia coli chemistry, RNA, Ribosomal, 5S chemistry
- Abstract
Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA. Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE. Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility. We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage. We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage. Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation. Furthermore, combined with the results from recent analyses of E. coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E. coli mRNA. The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
17. The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.
- Author
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Kaberdin VR, Miczak A, Jakobsen JS, Lin-Chao S, McDowall KJ, and von Gabain A
- Subjects
- Base Sequence, Conserved Sequence, DNA Primers genetics, Endoribonucleases chemistry, Endoribonucleases metabolism, Macromolecular Substances, RNA Processing, Post-Transcriptional, RNA, Bacterial metabolism, RNA, Messenger metabolism, RNA, Ribosomal metabolism, Species Specificity, Substrate Specificity, Cyanobacteria enzymology, Cyanobacteria genetics, Endoribonucleases genetics, Escherichia coli enzymology, Escherichia coli genetics, Evolution, Molecular
- Abstract
Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.
- Published
- 1998
- Full Text
- View/download PDF
18. RNA components of Escherichia coli degradosome: evidence for rRNA decay.
- Author
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Bessarab DA, Kaberdin VR, Wei CL, Liou GG, and Lin-Chao S
- Subjects
- Chromatography, Affinity, Endoribonucleases immunology, Endoribonucleases isolation & purification, Escherichia coli enzymology, Multienzyme Complexes immunology, Multienzyme Complexes isolation & purification, Oligopeptides, Peptides, Polyribonucleotide Nucleotidyltransferase immunology, Polyribonucleotide Nucleotidyltransferase isolation & purification, RNA Precursors metabolism, RNA, Ribosomal, 16S metabolism, RNA, Ribosomal, 23S metabolism, RNA, Ribosomal, 5S metabolism, RNA, Transfer metabolism, Substrate Specificity, Endoribonucleases metabolism, Multienzyme Complexes metabolism, Polyribonucleotide Nucleotidyltransferase metabolism, RNA Helicases, RNA Processing, Post-Transcriptional, RNA, Bacterial metabolism, RNA, Ribosomal metabolism
- Abstract
Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.
- Published
- 1998
- Full Text
- View/download PDF
19. Characterization of the structure and melting behavior of the loop I fragment of ColE1 RNA I.
- Author
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Lin TH, Lin HD, Yang JL, Kaberdin VR, Lin-Chao S, and Huang TH
- Subjects
- Base Composition, Magnetic Resonance Spectroscopy, Nucleic Acid Conformation, Plasmids genetics, Temperature, RNA, Antisense chemistry
- Abstract
We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5'-end, and a 31-mer which has 11 5'-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5' end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32 degrees C and 71 degrees C clearly discernible. In conjunction with NMR melting study the major transition at 71 degrees C is assigned to the overall melting of the stem region and the 32 degrees C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.
- Published
- 1996
- Full Text
- View/download PDF
20. Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops.
- Author
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McDowall KJ, Kaberdin VR, Wu SW, Cohen SN, and Lin-Chao S
- Subjects
- Base Sequence, Endoribonucleases antagonists & inhibitors, Escherichia coli enzymology, Molecular Sequence Data, Mutation, Oligoribonucleotides metabolism, RNA, Bacterial chemistry, RNA, Bacterial genetics, Ribonuclease T1 metabolism, Substrate Specificity, Endoribonucleases metabolism, Nucleic Acid Conformation, RNA, Bacterial metabolism
- Abstract
The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.
- Published
- 1995
- Full Text
- View/download PDF
21. Use of oligonucleotides and nick translation for site-directed mutagenesis in plasmids.
- Author
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Drutsa VL and Kaberdin VR
- Subjects
- Base Sequence, Molecular Sequence Data, Cloning, Molecular methods, DNA, Single-Stranded genetics, Mutagenesis, Site-Directed genetics, Oligodeoxyribonucleotides genetics, Plasmids genetics
- Published
- 1992
- Full Text
- View/download PDF
22. [An effective method for site-directed mutagenesis in plasmids and cloning single-stranded DNA fragments].
- Author
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Drutsa VL, Kaberdin VR, Koroleva ON, and Shilov IA
- Subjects
- Autoradiography, Bacteriophages genetics, Base Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Genes, Viral, Molecular Sequence Data, Polymerase Chain Reaction, Terminator Regions, Genetic, Transcription, Genetic, DNA, Single-Stranded genetics, Mutagenesis, Site-Directed, Plasmids
- Abstract
A three primer variant of the earlier devised oligonucleotide-directed mutagenesis in plasmids is described, useful also for the fast cloning of single-stranded DNA products of the asymmetric polymerase chain reaction (PCR). Using this method for plasmid pHD-001-14-11, a 59 b. p. deletion and a 7 b. p. insertion were simultaneously introduced at 81% frequency, and the PCR-copied phage fd transcription terminator (26 b. p.) was inserted with the yield of 67%.
- Published
- 1991
23. [A simple way of introducing a transcription initiation signal in vector DNA].
- Author
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Drutsa VL, Kaberdin VR, and Koroleva ON
- Subjects
- Base Sequence, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Mutagenesis, Site-Directed, Operon, Plasmids, Promoter Regions, Genetic, DNA genetics, Transcription, Genetic
- Abstract
A new effective method of site-specific mutagenesis in the close vicinity of unique restriction sites of plasmids, based on the use of two oligonucleotide primers, mutagenic and auxiliary, has been suggested. A site-specific insertion of Pribnow box (TATAATG) before promoterless gal operon of the promoter-testing plasmid pHD-001-14-11 has been performed with the yield of mutants up to 95%. Data on the gal operon expression and S1-nuclease mapping of the transcription start point indicate the formation of an active promoter in the region of the insertion.
- Published
- 1991
24. Simple approach for oligonucleotide-directed mutagenesis of any double-stranded circular DNA.
- Author
-
Drutsa VL and Kaberdin VR
- Subjects
- Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, DNA, Circular isolation & purification, Exodeoxyribonucleases, Genetic Techniques, Molecular Sequence Data, Plasmids, Restriction Mapping, DNA, Circular genetics, Mutagenesis, Site-Directed
- Abstract
A new site-directed method for introducing mutations into any region of plasmid vector close to the unique restriction site is described. It is based on the use of 5'-phosphorylated mutagenic and nonphosphorylated auxiliary oligonucleotides and a specific combination of enzymatic procedures including 'nick-translation' as a key step. The method efficiency was demonstrated by constructing the deletion-insertion mutation which creates the consensus Pribnow box in a promoter-testing plasmid. The yield of the target mutation was up to 85-95%.
- Published
- 1991
- Full Text
- View/download PDF
25. General method for site-directed mutagenesis and cloning of synthetic single-stranded DNA in circular vectors.
- Author
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Drutsa VL and Kaberdin VR
- Subjects
- Bacteriophage T4 enzymology, DNA Ligases metabolism, DNA-Directed DNA Polymerase metabolism, Exodeoxyribonucleases, Genetic Vectors, Cloning, Molecular methods, DNA, Single-Stranded chemical synthesis, DNA, Single-Stranded genetics, Mutagenesis, Site-Directed
- Published
- 1991
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