Struktur-Funtionsbeziehung in den spleißosomalen Proteinen des U4/U6•U5 tri-snRNP

Abstract

Subject of this thesis is the splicesosmal U5-particle. The functional relationship of the proteins is investigated by means of limited proteolysis and spectroscopy. Due to the splicesosome's dynamic nature and its heterogen composition, it is not yet possible to achieve a highly resolved structure of the whole spliceosome.The basis for a promising structural analysis is the definition of the smallest interacting area. The first step of this task is the generation and analysis of crystallisable amounts of the protein components, which has been carried out in this work. Several different ways of protein production are possible and have been tried out with the following results: Firstly, the bacterial expression of human U5-specific proteins is not a promising approach, since bacterial cells do not posses the required chaperones, membranes or enzymes for protein-modification. Secondly, the production of spliceosomal proteins in yeast cells is not large enough for structural analysis. Thirdly, however, from HeLa nuclear extracts human U5-snRNP can be successfully prepared, which, in turn, can be used to gain sufficient amounts of a heterotetrameric sub-complex for the required preparatory work of defining the smallest interacting area and, eventually, crystallisation experiments. The latter has been done for the first time in course of this work. Through the data gathered from those limited proteolysis experiments a core-particle was defined which has potential for further crystallisation experiments.In order to solve further problems, like heterologous expression of particle-specific human proteins, an insect cell system was established. The possibility to express interacting domains of the U5-specific proteins and their full-length constructs had not been given before. The data acquired by and described in this work enables the reconstitution of the interacting core and its biochemical characterisation as well as future structural analysis.