Identification of basepairs within Tn5 termini that are critical sfor H-NS binding to the transpososome and regulation of Tn5 transposition

<p>Abstract</p> <p>Background</p> <p>The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly <it>in vitro </it>and disruption of the <it>hns </it>gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition <it>in vivo </it>through an interaction with the transposition machinery as disruption of the <it>hns </it>gene has pleiotropic effects on <it>Escherichia coli</it>, the organism used in this study.</p> <p>Results</p> <p>In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition <it>in vivo </it>by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation.</p> <p>Conclusions</p> <p>H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.</p

Implications for Selves and Students Through Introducing New Pedagogical Strategies Into Our Teacher Education Practice

Collaborative self-study involving three mid-career teacher educators at different universities who introduced three new teaching strategies into their courses. Strategies promoted students to actively work with one another to construct and share their understandings. Researchers investigated implications for self, practices, outcomes, and relationships with students. Data included researchers’ reflective journals, Zoom conferences, course materials, teaching notes, and students’ survey responses and feedback. Each instructor’s students were encouraged to critique and reflect on the strategies’ utilization. Four themes emerged: unsettling consequences of change, renewed energy and enthusiasm for teaching, new skills and perspectives, and perceptions of student engagement, learning, and relationships

H-NS binds with high affinity to the Tn10 transpososome and promotes transpososome stabilization

H-NS is a bacterial DNA-binding protein that regulates gene expression and DNA transposition. In the case of Tn10, H-NS binds directly to the transposition machinery (i.e. the transpososome) to influence the outcome of the reaction. In the current work we evaluated the binding affinity of H-NS for two forms of the Tn10 transpososome, including the initial folded form and a pre-unfolded form. These two forms differ in that IHF is bound to the former but not the latter. IHF binding induces a bend (or fold) in the transposon end that facilitates transpososome formation. However, the continued presence of IHF in the transpososome inhibits intermolecular transposition events. We show that H-NS binds particularly strongly to the pre-unfolded transpososome with an apparent Kd of ∼0.3 nM. This represents the highest affinity interaction between H-NS and a binding partner documented to date. We also show that binding of H-NS to the transpososome stabilizes this structure and propose that both high-affinity binding and stabilization result from the combined interaction between H-NS and DNA and H-NS and transposase within the transpososome. Mechanistic implications for tight binding of H-NS to the transpososome and transpososome stabilization are considered

Interrogating the Generalizability of Portfolio Assessments of Beginning Teachers: A Qualitative Study

This qualitative study is intended to illuminate factors that affect the generalizability of portfolio assessments of beginning teachers. By generalizability, we refer here to the extent to which the portfolio assessment supports generalizations from the particular evidence reflected in the portfolio to the conception of competent teaching reflected in the standards on which the assessment is based. Or, more practically, “The key question is, ‘How likely is it that this finding would be reversed or substantially altered if a second, independent assessment of the same kind were made?’” (Cronbach, Linn, Brennan, and Haertel, 1997, p. 1). In addressing this question, we draw on two kinds of evidence that are rarely available: comparisons of two different portfolios completed by the same teacher in the same year and comparisons between a portfolio and a multi-day case study (observation and interview completed shortly after portfolio submission) intended to parallel the evidence called for in the portfolio assessment. Our formative goal is to illuminate issues that assessment developers and users can take into account in designing assessment systems and appropriately limiting score interpretations

The Effect of Mobile Element IS10 on Experimental Regulatory Evolution in Escherichia coli

Mobile genetic elements are widespread in bacteria, where they cause several kinds of mutations. Although their effects are on the whole negative, rare beneficial mutations caused by insertion sequence elements are frequently selected in some experimental evolution systems. For example, in earlier work, we found that strains of Escherichia coli that lack the sigma factor RpoS adapt to a high-osmolarity environment by the insertion of element IS10 into the promoter of the otsBA operon, rewiring expression from RpoS dependent to RpoS independent. We wished to determine how the presence of IS10 in the genome of this strain shaped the evolutionary outcome. IS10 could influence the outcome by causing mutations that confer adaptive phenotypes that cannot be achieved by strains without the element. Alternatively, IS10 could influence evolution by increasing the rate of appearance of certain classes of beneficial mutations even if they are no better than those that could be achieved by a strain without the element. We found that populations evolved from an IS10-free strain did not upregulate otsBA. An otsBA-lacZY fusion facilitated the recovery of a number of mutations that upregulate otsB without involving IS10 and found that two caused greater fitness increases than IS10 insertion, implying that evolution could have upregulated otsBA in the IS10-free strain. Finally, we demonstrate that there is epistasis between the IS10 insertion into the otsBA promoter and the other adaptive mutations, implying that introduction of IS10 into the otsBA promoter may alter the trajectory of adaptive evolution. We conclude that IS10 exerts its effect not by creating adaptive phenotypes that could not otherwise occur but by increasing the rate of appearance of certain adaptive mutations

Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

A bifunctional DNA binding region in Tn5 transposase

© 2008 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License 2.5. The definitive version was published in Molecular Microbiology 67 (2008): 528-540, doi:10.1111/j.1365-2958.2007.06056.x.Tn5 transposition is a complicated process that requires the formation of a highly ordered protein–DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein–DNA contacts, although many protein–DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase–DNA binding events.This work was supported by the NIH [GM50693], the University of Wisconsin at Madison [WIS04792], and through the Evelyn Mercer Professorship in Biochemistry and Molecular Biology

Alternative Mechanisms for Tn5 Transposition

Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism. The behavior of transposon Tn5 is anomalous. In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism. To explain this anomaly, a model is presented in which the two mechanisms are not independent—as widely believed—but could represent alternate outcomes of a common transpositional pathway

Carcinoembryonic Antigen Gene Family

The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin supergene family and can be divided into two main subgroups based on sequence comparisons. In humans it is clustered on the long arm of chromosome 19 and consists of approximately 20 genes. The CEA subgroup genes code for CEA and its classical crossreacting antigens, which are mainly membrane-bound, whereas the other subgroup genes encode the pregnancy-specific glycoproteins (PSG), which are secreted. Splice variants of individual genes and differential post-translational modifications of the resulting proteins, e.g., by glycosylation, indicate a high complexity in the number of putative CEA-related molecules. So far, only a limited number of CEA-related antigens in humans have been unequivocally assigned to a specific gene. Rodent CEA-related genes reveal a high sequence divergence and, in part, a completely different domain organization than the human CEA gene family, making it difficult to determine individual gene counterparts. However, rodent CEA-related genes can be assigned to human subgroups based on similarity of expression patterns, which is characteristic for the subgroups. Various functions have been determined for members of the CEA subgroup in vitro, including cell adhesion, bacterial binding, an accessory role for collagen binding or ecto-ATPases activity. Based on all that is known so far on its biology, the clinical outlook for the CEA family has been reassessed