Structure and Function of a Ribonucleoprotein Particle
The Kothe research group studies the structure and function of small ribonucleoproteins which are involved in ribosome biogenesis, in particular in modification of ribosomal RNA, with the aim of better understanding how the cell assembles large RNAprotein complexes such as the ribosome.
My basic research investigates the mechanism of the early stages in ribosome biogenesis on a molecular level. For this aim, I am applying a powerful, multidisciplinary combination of traditional yeast genetics and molecular biology with state-of-the-art biochemistry and biophysics using bacterial, archaeal and eukaryotic model systems.
The Globalink student will assist in all stages of the project allowing him to get detailed experience in many areas of biochemistry and molecular biology. One of our main questions addresses the function of certain structural elements of the ribonucleoprotein particle for example for substrate recognition or complex formation. To answer this question, we alter specific protein or RNA regions using site-directed mutagenesis methods. During his/her internship, the Globalink student will use these techniques to generate mutations in the DNAs encoding the protein components of the ribonucleoproteins complex. In order to reconstitute the mutant ribonucleoprotein particle, the mutant protein needs to be recombinantly overexpressed in E. coli and subsequently be purified by affinity and other types of chromatography.
Also the other proteins of the ribonucleoproteins complex will be purified by similar methods. The success of the protein purifications is followed by SDS-PAGE. Next, the RNA component of the ribonucleoproteins complex is generated by in vitro transcription followed by purification using anion exchange chromatography which is monitored by RNA-Urea-PAGE. Lastly, the
student will analyze formation of the ribonucleoprotein complex by size-exclusion chromatography comparing both the wild-type and the mutant components. Together with my Ph.D. student, the Globalink student will also have the opportunity to assess effects of the introduced mutation on the ability of the ribonucleoproteins particle to modify RNA. In summary, this project aims at furthering our understanding of ribonucleoprotein structure and function in ribosome biogenesis. This knowledge will be important to target this critical cellular pathway in different diseases.
