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Projekt
Functional characterization of the chaperone network connected to the eucaryotic ribosome
Antragstellerin
Professorin Dr. Sabine Karola Rospert
Fachliche Zuordnung
Biochemie
Förderung
Förderung von 2008 bis 2014
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 50070218
It is now recognized that ribosome-bound nascent peptides can interact with components of the polypeptide exit tunnel, acquire secondary structure, and transmit information to distant sites of the ribosome. The underlying mechanisms, however, are only incompletely understood, specifically in the eukaryotic system. Aim of the project is to understand how the interplay between components of the eukaryotic ribosome, nascent chains, and ribosomebound protein biogenesis factors (RPBs) affects the early steps of protein biogenesis. We will focus on the analysis of the ribosomal proteins Rpl17, Rpl4, and Rpl39, which contact the interior of the tunnel in eukaryotes. Random mutagenesis within Rpl17 and Rpl4 will be employed to generate a set of conditionally lethal yeast mutants. Yeast strains expressing mutant versions of Rpl17, Rpl4, or lacking Rpl39 will be characterized in vivo. We will focus on protein targeting and folding defects in the mutant strains. An array of biochemical methods will then be employed to compare the fate of nascent chains bound to wild type and mutant ribosomes. First, we will determine by what mechanism the ribosomal tunnel proteins affect nascent chain folding. Second, we will analyze if nascent chain folding impacts on the function of the major ribosome-bound protein biogenesis factors, SRP, NAC, and Ssb. In particular we plan to trace the transfer of a signal anchor sequence from the tunnel to SRP. The analysis shall unravel if intraribosomal folding of a signal anchor sequence is a prerequisite for SRP-dependent targeting, and what exactly defines SRPdepended substrates. Finally, we will employ the system to test the hypothesis that nascent chains can facilitate structural changes within the ribosomal binding site(s) of RPBs from within the ribosomal tunnel.
DFG-Verfahren
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