The Experts below are selected from a list of 138 Experts worldwide ranked by ideXlab platform
David B. Haniford - One of the best experts on this subject based on the ideXlab platform.
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Transposons Tn10 and Tn5
Microbiology spectrum, 2015Co-Authors: David B. Haniford, Michael J EllisAbstract:The study of the bacterial transposons Tn10 and Tn5 has provided a wealth of information regarding steps in nonreplicative DNA transposition, transpososome dynamics and structure, as well as mechanisms employed to regulate transposition. The focus of ongoing research on these transposons is mainly on host regulation and the use of the Tn10 antisense system as a platform to develop riboregulators for applications in synthetic biology. Over the past decade two new regulators of both Tn10 and Tn5 transposition have been identified, namely H-NS and Hfq proteins. These are both global regulators of gene expression in enteric bacteria with functions linked to stress-response pathways and virulence and potentially could link the Tn10 and Tn5 systems (and thus the transfer of antibiotic resistance genes) to environmental cues. Work summarized here is consistent with the H-NS protein working directly on transposition complexes to upregulate both Tn10 and Tn5 transposition. In contrast, evidence is discussed that is consistent with Hfq working at the level of transposase expression to downregulate both systems. With regard to Tn10 and synthetic biology, some recent work that incorporates the Tn10 antisense RNA into both transcriptional and translational riboswitches is summarized.
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Tn10 is10 transposition is downregulated at the level of transposase expression by the rna binding protein hfq
Molecular Microbiology, 2010Co-Authors: Joseph A Ross, Simon J. Wardle, David B. HanifordAbstract:Summary We show in this work that disruption of the hfq gene in Escherichia coli causes a large increase in IS10 transposition when IS10 is present on a multi-copy plasmid. Hfq is an RNA-binding protein that regulates the expression of a large number of genes at the post-transcriptional level by promoting the pairing of mRNAs with partially complementary short RNAs. As the translation of IS10 transposase mRNA (RNA-IN) is inhibited by an IS10-encoded anti-sense RNA (RNA-OUT), it seemed likely that Hfq would negatively regulate Tn10/IS10 transposition by promoting anti-sense inhibition of RNA-IN translation. Consistent with this, we show that Hfq promotes pairing of RNA-IN and RNA-OUT in vitro and downregulates RNA-IN expression in vivo. However, we also show that Hfq negatively regulates Tn10 transposition when no functional anti-sense RNA is produced. Taken together, the results suggest that Hfq acts at two distinct steps to inhibit Tn10/IS10 transposition. This is the first example of Hfq regulating a bacterial transposition reaction.
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Tn10/IS10 transposition is downregulated at the level of transposase expression by the RNA‐binding protein Hfq
Molecular microbiology, 2010Co-Authors: Joseph A Ross, Simon J. Wardle, David B. HanifordAbstract:Summary We show in this work that disruption of the hfq gene in Escherichia coli causes a large increase in IS10 transposition when IS10 is present on a multi-copy plasmid. Hfq is an RNA-binding protein that regulates the expression of a large number of genes at the post-transcriptional level by promoting the pairing of mRNAs with partially complementary short RNAs. As the translation of IS10 transposase mRNA (RNA-IN) is inhibited by an IS10-encoded anti-sense RNA (RNA-OUT), it seemed likely that Hfq would negatively regulate Tn10/IS10 transposition by promoting anti-sense inhibition of RNA-IN translation. Consistent with this, we show that Hfq promotes pairing of RNA-IN and RNA-OUT in vitro and downregulates RNA-IN expression in vivo. However, we also show that Hfq negatively regulates Tn10 transposition when no functional anti-sense RNA is produced. Taken together, the results suggest that Hfq acts at two distinct steps to inhibit Tn10/IS10 transposition. This is the first example of Hfq regulating a bacterial transposition reaction.
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the nucleoid binding protein h ns acts as an anti channeling factor to favor intermolecular Tn10 transposition and dissemination
Journal of Molecular Biology, 2008Co-Authors: Randeep K. Singh, Simon J. Wardle, Janine Liburd, David B. HanifordAbstract:Dissemination of the bacterial transposon Tn10 is limited by target site channeling, a process wherein the transposon ends are forced to interact with and insert into a target site located within the transposon. Integration host factor (IHF) promotes this self-destructive event by binding to the transpososome and forming a DNA loop close to one or both transposon ends; this loop imposes geometric and topological constraints that are responsible for channeling. We demonstrate that a second 'host' protein, histone-like nucleoid structuring protein (H-NS), acts as an anti-channeling factor to limit self-destructive intramolecular transposition events in vitro. Evidence that H-NS competes with IHF for binding to the Tn10 transpososome to block channeling and that this event is relatively insensitive to the level of DNA supercoiling present in the Tn10-containing substrate plasmid are presented. This latter observation is atypical for H-NS, as H-NS binding to other DNA sequences, such as promoters, is generally affected by subtle changes in DNA structure.
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The global regulator H-NS binds to two distinct classes of sites within the Tn10 transpososome to promote transposition.
Molecular microbiology, 2007Co-Authors: Christopher Ward, Simon J. Wardle, Randeep K. Singh, David B. HanifordAbstract:The histone-like nucleoid structuring protein (H-NS) is a global transcriptional regulator that influences stress response and virulence pathways in Gram-negative bacteria. H-NS also promotes Tn10 transposition by binding directly to the transpososome and inducing a conformational change in the transpososome that favours intermolecular transposition events. H-NS binds preferentially to curved DNA and can bend non-curved DNA, it self-oligomerizes and can interact with other proteins. To determine what functions of H-NS are important in promoting Tn10 transposition, we have examined the ability of two mutant forms of H-NS, P116S and 1-64, to act in Tn10 transposition. We provide evidence that the initial interaction of H-NS with the transpososome is dependent on H-NS binding to a specific structure in DNA flanking the transposon end. Additional molecules of H-NS then bind within the transposon end. This latter event appears to be directed by H-NS binding to the Tn10 transposase protein, and is important in maintaining the transpososome in a conformation that promotes intermolecular transposition. The binding of H-NS to a transposase protein is a novel function for this important regulatory molecule.
Nancy Kleckner - One of the best experts on this subject based on the ideXlab platform.
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Tn10 Transposition via a DNA Hairpin Intermediate
Cell, 1998Co-Authors: Angela K Kennedy, Nancy Kleckner, Anjan Guhathakurta, David B. HanifordAbstract:Abstract We present evidence that excision of the nonreplicative transposon Tn10 involves three distinct chemical steps, first-strand nicking, hairpin formation, and hairpin resolution. This three-step mechanism makes it possible for a single protein-active site to cleave two DNA strands of opposite polarity, as appears to be the case in this reaction. We infer the existence of alternating bifunctionality within the active site with suitable modulation of substrate components between steps. DNA double-strand breaks are also made by a "hairpin mechanism" in V(D)J recombination, possibly reflecting the same basic constraints faced in the Tn10 system. Similarities in the basic chemical steps in Tn10 transposition and V(D)J recombination suggest that the V(D)J mechanism may have evolved from a bacterial transposition system.
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The Tn10 Synaptic Complex Can Capture a Target DNA only after Transposon Excision
Cell, 1997Co-Authors: Janice Sakai, Nancy KlecknerAbstract:Abstract Tn10 transposes nonreplicatively. Staged in vitro reactions demonstrate that a Tn10 synaptic complex can become committed to a particular target DNA molecule via a noncovalent interaction in the absence of strand transfer. Commitment occurs only after double-strand cleavage at both transposon ends (in "double-end break" [DEB] complexes). Stable noncovalent DEB–target DNA cocomplexes can be detected, but no cocomplexes occur with synaptic complexes containing uncleaved ends. Preincubation of DEB complexes with target DNA accelerates the rate of strand transfer. Postcleavage target capture is remarkable for Tn10; Mu and Tn7 select a target site prior to cleavage. Promiscuous target selection may favor evolution of IS-based composite elements while being suicidal for other types of transposons.
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IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.
The EMBO journal, 1996Co-Authors: R. M. Chalmers, Nancy KlecknerAbstract:Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.
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is10 Tn10 transposition efficiently accommodates diverse transposon end configurations
The EMBO Journal, 1996Co-Authors: R. M. Chalmers, Nancy KlecknerAbstract:Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.
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Tn10 and IS10 Transposition and Chromosome Rearrangements: Mechanism and Regulation In Vivo and In Vitro
Current topics in microbiology and immunology, 1996Co-Authors: Nancy Kleckner, R. M. Chalmers, Douglas S. Kwon, J. Sakai, S. BollandAbstract:Tn10 is a composite transposon. It comprises a pair of IS10 insertion sequences located in opposite orientation flanking ~6.7 kb of unique sequences; these unique sequences encode a tetracycline resistance determinant and other determinants whose functions remain to be identified (Fig. 1A; Kleckner 1989). One of Tn10’s two IS10 elements, IS10-Right, is structurally and functionally intact and is considered to be the “wild type” IS10. IS10 encodes a single transposase protein which mediates transposition by interacting with specific sequences at two oppositely oriented IS10 (or Tn10) termini. The termini of IS10 are subtly different and are referred to as the “outside” and “inside” end, respectively, by virtue of their position in Tn10. IS10-Left is structurally intact but encodes a substantially defective transposase.
R. M. Chalmers - One of the best experts on this subject based on the ideXlab platform.
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is10 Tn10 transposition efficiently accommodates diverse transposon end configurations
The EMBO Journal, 1996Co-Authors: R. M. Chalmers, Nancy KlecknerAbstract:Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.
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IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.
The EMBO journal, 1996Co-Authors: R. M. Chalmers, Nancy KlecknerAbstract:Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.
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Tn10 and IS10 Transposition and Chromosome Rearrangements: Mechanism and Regulation In Vivo and In Vitro
Current topics in microbiology and immunology, 1996Co-Authors: Nancy Kleckner, R. M. Chalmers, Douglas S. Kwon, J. Sakai, S. BollandAbstract:Tn10 is a composite transposon. It comprises a pair of IS10 insertion sequences located in opposite orientation flanking ~6.7 kb of unique sequences; these unique sequences encode a tetracycline resistance determinant and other determinants whose functions remain to be identified (Fig. 1A; Kleckner 1989). One of Tn10’s two IS10 elements, IS10-Right, is structurally and functionally intact and is considered to be the “wild type” IS10. IS10 encodes a single transposase protein which mediates transposition by interacting with specific sequences at two oppositely oriented IS10 (or Tn10) termini. The termini of IS10 are subtly different and are referred to as the “outside” and “inside” end, respectively, by virtue of their position in Tn10. IS10-Left is structurally intact but encodes a substantially defective transposase.
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Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.
The EMBO journal, 1995Co-Authors: J. Sakai, R. M. Chalmers, Nancy KlecknerAbstract:The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.
Simon J. Wardle - One of the best experts on this subject based on the ideXlab platform.
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Tn10 is10 transposition is downregulated at the level of transposase expression by the rna binding protein hfq
Molecular Microbiology, 2010Co-Authors: Joseph A Ross, Simon J. Wardle, David B. HanifordAbstract:Summary We show in this work that disruption of the hfq gene in Escherichia coli causes a large increase in IS10 transposition when IS10 is present on a multi-copy plasmid. Hfq is an RNA-binding protein that regulates the expression of a large number of genes at the post-transcriptional level by promoting the pairing of mRNAs with partially complementary short RNAs. As the translation of IS10 transposase mRNA (RNA-IN) is inhibited by an IS10-encoded anti-sense RNA (RNA-OUT), it seemed likely that Hfq would negatively regulate Tn10/IS10 transposition by promoting anti-sense inhibition of RNA-IN translation. Consistent with this, we show that Hfq promotes pairing of RNA-IN and RNA-OUT in vitro and downregulates RNA-IN expression in vivo. However, we also show that Hfq negatively regulates Tn10 transposition when no functional anti-sense RNA is produced. Taken together, the results suggest that Hfq acts at two distinct steps to inhibit Tn10/IS10 transposition. This is the first example of Hfq regulating a bacterial transposition reaction.
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Tn10/IS10 transposition is downregulated at the level of transposase expression by the RNA‐binding protein Hfq
Molecular microbiology, 2010Co-Authors: Joseph A Ross, Simon J. Wardle, David B. HanifordAbstract:Summary We show in this work that disruption of the hfq gene in Escherichia coli causes a large increase in IS10 transposition when IS10 is present on a multi-copy plasmid. Hfq is an RNA-binding protein that regulates the expression of a large number of genes at the post-transcriptional level by promoting the pairing of mRNAs with partially complementary short RNAs. As the translation of IS10 transposase mRNA (RNA-IN) is inhibited by an IS10-encoded anti-sense RNA (RNA-OUT), it seemed likely that Hfq would negatively regulate Tn10/IS10 transposition by promoting anti-sense inhibition of RNA-IN translation. Consistent with this, we show that Hfq promotes pairing of RNA-IN and RNA-OUT in vitro and downregulates RNA-IN expression in vivo. However, we also show that Hfq negatively regulates Tn10 transposition when no functional anti-sense RNA is produced. Taken together, the results suggest that Hfq acts at two distinct steps to inhibit Tn10/IS10 transposition. This is the first example of Hfq regulating a bacterial transposition reaction.
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the nucleoid binding protein h ns acts as an anti channeling factor to favor intermolecular Tn10 transposition and dissemination
Journal of Molecular Biology, 2008Co-Authors: Randeep K. Singh, Simon J. Wardle, Janine Liburd, David B. HanifordAbstract:Dissemination of the bacterial transposon Tn10 is limited by target site channeling, a process wherein the transposon ends are forced to interact with and insert into a target site located within the transposon. Integration host factor (IHF) promotes this self-destructive event by binding to the transpososome and forming a DNA loop close to one or both transposon ends; this loop imposes geometric and topological constraints that are responsible for channeling. We demonstrate that a second 'host' protein, histone-like nucleoid structuring protein (H-NS), acts as an anti-channeling factor to limit self-destructive intramolecular transposition events in vitro. Evidence that H-NS competes with IHF for binding to the Tn10 transpososome to block channeling and that this event is relatively insensitive to the level of DNA supercoiling present in the Tn10-containing substrate plasmid are presented. This latter observation is atypical for H-NS, as H-NS binding to other DNA sequences, such as promoters, is generally affected by subtle changes in DNA structure.
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The global regulator H-NS binds to two distinct classes of sites within the Tn10 transpososome to promote transposition.
Molecular microbiology, 2007Co-Authors: Christopher Ward, Simon J. Wardle, Randeep K. Singh, David B. HanifordAbstract:The histone-like nucleoid structuring protein (H-NS) is a global transcriptional regulator that influences stress response and virulence pathways in Gram-negative bacteria. H-NS also promotes Tn10 transposition by binding directly to the transpososome and inducing a conformational change in the transpososome that favours intermolecular transposition events. H-NS binds preferentially to curved DNA and can bend non-curved DNA, it self-oligomerizes and can interact with other proteins. To determine what functions of H-NS are important in promoting Tn10 transposition, we have examined the ability of two mutant forms of H-NS, P116S and 1-64, to act in Tn10 transposition. We provide evidence that the initial interaction of H-NS with the transpososome is dependent on H-NS binding to a specific structure in DNA flanking the transposon end. Additional molecules of H-NS then bind within the transposon end. This latter event appears to be directed by H-NS binding to the Tn10 transposase protein, and is important in maintaining the transpososome in a conformation that promotes intermolecular transposition. The binding of H-NS to a transposase protein is a novel function for this important regulatory molecule.
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The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition
Genes & development, 2005Co-Authors: Simon J. Wardle, Michelle O'carroll, Keith M. Derbyshire, David B. HanifordAbstract:The histone-like nucleoid structuring (H-NS) protein is a global transcriptional regulator that is known to regulate stress response pathways and virulence genes in bacteria. It has also been implicated in the regulation of bacterial transposition systems, including Tn10. We demonstrate here that H-NS promotes Tn10 transposition by binding directly to the transposition complex (or transpososome). We present evidence that, upon binding, H-NS induces the unfolding of the Tn10 transpososome and helps to maintain the transpososome in an unfolded state. This ensures that intermolecular (as opposed to self-destructive intramolecular) transposition events are favored. We present evidence that H-NS binding to the flanking donor DNA of the transpososome is the initiating event in the unfolding process. We propose that by recruiting H-NS as a modulator of transposition, Tn10 has evolved a means of sensing changes in host physiology, as the amount of H-NS in the cell, as well its activity, are responsive to changes in environmental conditions. Sensing of environmental changes through H-NS would allow transposition to occur when it is most opportune for both the transposon and the host.
S. Bolland - One of the best experts on this subject based on the ideXlab platform.
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Tn10 and IS10 Transposition and Chromosome Rearrangements: Mechanism and Regulation In Vivo and In Vitro
Current topics in microbiology and immunology, 1996Co-Authors: Nancy Kleckner, R. M. Chalmers, Douglas S. Kwon, J. Sakai, S. BollandAbstract:Tn10 is a composite transposon. It comprises a pair of IS10 insertion sequences located in opposite orientation flanking ~6.7 kb of unique sequences; these unique sequences encode a tetracycline resistance determinant and other determinants whose functions remain to be identified (Fig. 1A; Kleckner 1989). One of Tn10’s two IS10 elements, IS10-Right, is structurally and functionally intact and is considered to be the “wild type” IS10. IS10 encodes a single transposase protein which mediates transposition by interacting with specific sequences at two oppositely oriented IS10 (or Tn10) termini. The termini of IS10 are subtly different and are referred to as the “outside” and “inside” end, respectively, by virtue of their position in Tn10. IS10-Left is structurally intact but encodes a substantially defective transposase.