Abstract
(English)
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The SRP network combines structural and functional methods to advance understanding of the SRP-mediated targeting process of membrane and secretory proteins to the endoplasmic reticulum (ER). Our focus has been the structure-function analysis of the SRP Alu-domain which is essential for the translation arrest activity of SRP. This function enhances the efficiency of ER translocation. In summary:
· In collaboration with the structure group of Dr. S. Cusack in Grenoble, we have determined high resolution structures of Alu-domain components. Our results indicate that formation of the SRP9/14-Alu RNA complex is a two-step process The proteins binds strongly to the evolutionary conserved 5' core and more weakly to the central stem region of the Alu portion of SRP RNA. We propose that the reversible second binding event is an essential feature in the mechanism of retarding ribosomal elongation.
· In functional studies, we identified a short conserved basic region in the C-terminal portion of SRP14 (amino acid residues 95-100) to be critical for elongation arrest. The final refinement in these studies has been greatly aided by the availability of the crystal structure.
· In a collaborative effort with the group of Dr. Dobberstein in Heidelberg, we have engaged in the identification of ribosomal proteins which are in proximity of the Alu-domain during the elongation cycle. We have observed four different proteins that became cross-linked to SRP14 in SRP-ribosome-nascent chain complexes. We showed that these proteins are ribosomal proteins which are associated with the large (three) or the small (1) subunit. Interestingly, two of the cross-links are only observed in the presence of a signal sequence in the nascent chain. This observation could be indicative of a structural rearrangement of SRP upon signal sequence recognition. We are in the process of identifying these proteins.
Weichenrieder, O., Stehlin, C., Kapp, U., Birse, D. E., Timmins, P. A., Strub, K., and Cusack, S. (2001). Hierarchical assembly of the Alu domain of the mammalian signal recognition particle. Rna 7, 731-40.
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