This research will continue, in several new directions, the present research program with the goal of gaining a detailed understanding of the molecular mechanisms of the transposition process, and its control, specifically of insertion sequence IS1. Insights gained to date put us in a position to answer very specific questions about the essential conponents of the transposition complex, the control of gene expression in IS1, and the regulation of transposition itself. The program of research has the additional long range goal of gaining a mechanistic understanding of the full range of recombinational events induced by the insertion sequence IS1, the molecular processes by which they occur, and the mechanisms for their regulation. Several basic molecular processes that are central to IS1 transposition promise to be novel and important. Among these are the set of key molecular events in transposition and control of gene expression that take place at the ends of IS1 (all within about 30 base-pairs), and the possibility that translational frameshifting is a critical regulatory event that takes place in the center of IS1. These mechanisms, appear to be different from those of other well-studied transposable elements like TnlO, Tn3, Tn7, Mu phage, IS5 and IS50. IS1 is one of the most widespread of transposable elements, and elucidation of the basic molecular mechanisms is essential to an understanding of the IS-mediated assembly of genes into plasmids and dissemination of genetic information in the bacterial world. The proposed project aims to study the apparent frameshifting that produces a fused InsA-B protein, which may be the ISI transposase. All the IS1-encoded components of the transposition complex, essential or accessory, will be characterized. The binding properties of the repressor (InsA) and the (putative) transposase will be studied via mutational analysis, of both the proteins and the DNA sites, and by physical methods. A new genetic system for the study of IS1 will be developed, which permits selection and screening for mutants in the transpostion pathway. Finally, new results with the fusion protein will be used to develop an in vitro system for IS1 transpostion, which will be used to dissect the process biochemically. Detailed molecular models of IS1 transpostion will be contructed from these results and tested further.
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