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Metrics details. The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7 , and insert just downstream of the highly conserved chromosomal glmS gene. In this study, we identified important nucleotides within this region that are crucial for TnsD- attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. Strikingly, whereas most transposons insert relatively randomly into many different sites, Tn 7 transposition is quite specific. When TnsD is the target selector, Tn 7 inserts at high frequency into a specific chromosomal site called an attachment site, attTn7 [ 4 ]. Insertion occurs directly downstream of the essential glmS gene, ensuring that it does not disrupt the glmS open reading frame and glmS expression is preserved [ 5 ].

Tnsd

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In this study, tnsd, we have made considerable progress in understanding the unique TnsD family of DNA-binding proteins by defining important protein tnsd for DNA binding and transposition, and key nucleotides for the TnsD- attTn7 interaction. As described in more detail below, we were unable to observe DNA binding in tnsd with various N-terminal fragments of TnsD, thus could not identify a discrete zinc finger motif-containing DNA-binding domain, tnsd. Yeast two-hybrid assay The yeast two-hybrid assay was performed according to the manufacturer's protocol Proquest Two-Hybrid System; Life Technologies Inc, tnsd.

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Tnsd

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites. This is a preview of subscription content, access via your institution.

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To be eligible for a return, your item must be in the same condition that you received it, factory sealed , with tags, and in its original packaging. The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7 , and insert just downstream of the highly conserved chromosomal glmS gene. Refund Policy. This may include the offer of fast shipping and easy returns, positive user ratings, and other factors. Lu F, Craig NL: Isolation and characterization of Tn 7 transposase gain-of-function mutants: a model for transposase activation. Previous work in vitro identified the 36 bp span of the TnsD binding site, and demonstrated that this sequence alone is sufficient to direct transposition [ 6 ]. The 36 bp TnsD-binding site lies within the 3' end of glmS , which encodes the C-terminus of the protein. In this study, we have further probed the interaction of TnsD with attTn7 and deepened our understanding of Tn7 site-specific insertion. Indeed, most of the DNA sequence divergence results from variation at the wobble position of each codon see below. Crosslinking between TnsD and attTn7. The interaction between full-length TnsC and TnsD is only slightly less than that of the robust interaction displayed by the Fos-Jun control, but equivalent to that of RB and E2F1 [ 22 ]. A suspension system in its single roller design features 3 or 4 thin cables eliminating the need for black drop — the black border above the projection screen. Binding reactions were performed with wild-type and mutant versions of TnsD proteins and wild-type TnsC protein as described previously [ 6 ].

Federal government websites often end in. The site is secure. The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7 , and insert just downstream of the highly conserved chromosomal glmS gene.

The minimal E. Gardner for Salmonella strains and P22 phages, and J. After hydroxylamine mutagenesis of TnsD and screening of 15, transformants, we isolated 16 TnsD mutants, 11 of which were missense mutants and five of which were nonsense mutants yielding truncated TnsD; only two mutants TnsD PL and TnsD RC were isolated twice Figure 4 , Figure 5 , Figure 6. Tn 7 inserts into attTn7 because TnsD specifically recognizes highly conserved sequences within the protein coding region of glmS [ 4 ], and recruits the rest of the transposition machinery to this site. Reactions were stopped by adjusting to 25 mM EDTA, followed by extraction with phenol:chloroform Steen H, Jensen ON: Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry. Zip code. Figure 7. Please get in touch if you have questions or concerns about your specific item. TnsC also interacts with and activates both subunits of the Tn 7 transposase TnsA and TnsB , which mediate breakage and joining of the ends of Tn 7 [ 25 ].