Mobile genetic elements have been found in a wide variety of procaryotic and eucaryotic organisms. They are widely thought to represent one of Nature's principal tools for the rearrangement of genetic material. Our long term goal is to understand in detail how the transposition of mobile genetic elements is controlled. As a step toward this goal, we will study the details of a novel regulatory mechanism in the bacterial insertion sequence IS10: the post-transcriptional control of IS10 transposase gene expression by a small IS10-specified anti-sense RNA. We term this control """"""""multicopy inhibition"""""""", and our working model is one where the anti-sense RNA binds to and inhibits translation of the transposase mRNA. This problem will be studied both genetically and biochemically. Using genetic means, we will introduce point mutations into IS10, and screen for mutants that are altered in multicopy inhibition. We expect to find two types: those that alter the quality and/or quantity of the anti-sense RNA, and those that alter the site, in the transposase mRNA, at which the anti-sense RNA acts. Mutants will be subjected to a battery of sophisticated genetic tests to determine how the mutations affect various aspects of IS10 behavior, including transposition, transposase function production, complementability, and multicopy inhibition. The transcriptional activity of these mutants, and the character of the transcripts they produce, will be studied in vivo, with particular attention given to the fate of these transcripts during multicopy inhibition. We will attempt to express and isolate large amounts of wild-type and mutant IS10 transcripts, to be used for in vitro studies on transcript binding. We will examine the hypothesis that multicopy inhibition is exerted at the level of translation of the IS10 transposase by studying translation in vitro, initially with the Weissbach di-peptide synthesis assay. By isolating and characterizing mutations in the E. coli host, we will attempt to determine which cellular processes participate in multicopy inhibition, and at what level. Particular emphasis will be placed on possible host functions that promote or retard pairing between complementary RNAs.
