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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 ].

Tnsd

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Tnsd

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. 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 ].

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However, when TnsC and TnsD were expressed simultaneously in the host cells, we observed a enhancement of lysogeny of nearly fold relative to cells expressing only TnsD. Steen H, Jensen ON: Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry. To determine directly whether TnsD binding to attTn7 is dependent on zinc, gel-shift assays were performed using wild-type TnsD and attTn7 in the presence of increasing amounts of the zinc chelator 1,phenanthroline. Correspondence to Nancy L Craig. Sorry, a shareable link is not currently available for this article. In this study, we identified important nucleotides within this region that are crucial for TnsD- attTn7 interaction. Sequencing of junctions of independent insertions from the in vivo transposition assay showed that Tn7 insertions were present in attTn7 in a sequence- and orientation-specific manner, with the 5-bp target duplication characteristic of Tn7 transposition, as occurs with wild-type TnsD data not shown. We also performed deletion analysis of TnsC using the yeast two-hybrid system Table 5. It has no homologs outside of Tn 7 -type transposons, about of which are now reported in Genbank. It seems likely that the DNA-mediated and protein-protein interactions are both important to TnsC-mediated activation of transposition.

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None of these iodouracil substitutions had a detectable effect on TnsD binding in vitro data not shown. Figure 2. We have now found, using a yeast two-hybrid assay, that TnsC and TnsD also interact directly in the absence of attTn7 [ 20 , 21 ] Table 5. Table 1 TnsD binding to attTn7 in vivo Full size table. Free and bound DNA are indicated for different positions along the attachment site. These assays show that TnsD also binds poorly to these mutant sites in vivo , supporting the view that the identity of these positions is important for the site-specific binding activity of TnsD. Projection Lenses Projectors Screens. Free Standard Shipping. We know from previous work that Tn7 transposition functions well in Salmonella [ 13 ]. Indeed, most of the DNA sequence divergence results from variation at the wobble position of each codon see below.