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. They are also viewed as """"""""selfish DNA"""""""" or molecular parasites that must co-exist with their hosts. The long term goal of this project is to understand in detail how the transposition of mobile genetic elements is controlled, and in what ways such elements have adapted to their hosts. As a step toward this goal, it is proposed to continue genetic and biochemical studies on 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 (alpha RNA). This alpha RNA pairs to its target, the transposase mRNA, in a sequence- dependent process, and the in vivo consequence of pairing is the endonucleolytic cleavage of both the alpha RNA and the mRNA by an unknown, host encoded ribonuclease. The biological significance of IS10 alpha RNA control is that it probably serves to limit the accumulation of IS10 elements in the cell. This is supported by experiments showing that alpha RNA control increases with increasing IS10 copy number. Considerable genetic and biochemical studies have already been accomplished on this regulatory system, now permitting more extensive analysis of the details of this control mechanism. The following specific goals are proposed. The structure of the alpha RNA and relevant regions of the mRNA will be determined, and the pairing process detailed. The structure of the paired species that is sensitive to endonucleolytic cleavage will also be identified. These studies will be augmented by ongoing genetic and biochemical analysis of the sequence specificity of pairing. A detailed analysis of the cleavage reaction will be conducted, in vivo as well as in a newly developed functional in vitro assay, and the endonuclease involved will be identified genetically and/or biochemically. The extent to which ribosomes are exclude from the paired species will be assessed, especially with regard to possible contribution to control in vivo. Host mutants in which alpha RNA control is perturbed (at several levels) have been isolated, and these will be further characterized, including isolation and characterization of the implicated genes and gene products.
