Architecture of the Tn7 Posttransposition Complex: An Elaborate Nucleoprotein Structure

JOURNAL OF MOLECULAR BIOLOGY

卷:401

Article

Holder, Jason W.¹  Craig, Nancy L.¹ 

[1] Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Dept Mol Biol & Genet, Baltimore, MD 21205 USA

关键词: transposition;    transposase;    nucleoprotein complex;    expressed protein ligation;    DNA footprinting;   

DOI  :  10.1016/j.jmb.2010.06.003

来源: Elsevier

【 摘 要 】

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Ins proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7::Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3' end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC. (C) 2010 Elsevier Ltd. All rights reserved.

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