| Most of the non-coding ('junk') DNA in eukaryotic genomes was created by non-LTR retrotransposons. In humans the continuous retrotransposition of L1 long interspersed elements (LINEs) and Alushort interspersed elements (SINEs) is responsible for approximately one quarter of genomic DNA. The autonomous (i.e. encoding reverse transcriptase) L1 elements are the major source of insertional mutagenesis, a potentially hazardous cellular process with fundamental evolutionary implications. The non-autonomous Alu elements are believed to efficiently exploit the L1 retrotransposition machinery for their own propagation. The mechanistic details of 'target-primed reverse transcription' (TPRT) by which the L1 proteins mediate target DNA cleavage and genomic re-insertion of associated RNA are unclear. It is equally obscure how Alu ribonucleoprotein particles (Alu RNPs) recruit L1 encoded protein(s) for genomic re-integration of Alu RNA. I propose to use X-ray crystallography to provide essential structural details on components and sub-complexes of the L1 retrotransposition machinery. In parallel, we will establish assays (cell-free and in vivo) to test how Alu RNPs interact with L1 protein(s). The immediate results should help to further position L1 elements - and non-LTR retrotransposons in general - in the evolutionary context of mobile genetic elements. They should also allow us to relate retrotransposition to processes like the action of telomerase, which contains a reverse transcriptase similar to the one encoded by L1. In the long run we would like to understand the chemical details of non-LTR retrotransposition as a whole and their significance for genome evolution. Furthermore, our data should provide ideas on whether and how to convert non-LTR retrotransposition into a genetic tool. |
