The objective of this research proposal is to advance the understanding of the basic process of protein transport across biological membranes. Our particular focus is on the signal recognition particle (SRP) as it plays a central role in secretion. A major component of all SRPs is an RNA molecule, regions of which are universally conserved. Our goal is to understand the structural and functional role of this RNA in the human system. We will define those regions of the human SRP RNA which are involved in particular aspects of SRP-mediated protein secretion and SRP assembly by using a combination of site-directed mutagenesis, site- directed cross-linking, and phylogenetic comparisons with cloned SRPcomponents. The major topics to be investigated in this proposal are: (1) The molecular mechanisms involved in signal recognition and protein translocation. SRPs reconstituted with mutant SRP RNAs will be used to test the translocation competence of secretory proteins in a cell-free translation-translocation system. The individual steps which are affected in the SRP-cycle, such as signal recognition, translation arrest, and release of the arrest, will be identified. (2) SRP assembly. Mutants of SRP RNA will be constructed by systematic site-directed mutagenesis, or selected after random mutagenesis, to study the interactions of the SRP proteins with the SRP RNA. Information about the binding sites on the proteins will be obtained by testing the ability of mutant polypeptides to interact with the SRP RNA or with partially assembled ribonucleoprotein particles (RNPs). Photochemical cross-linking with modified nucleotides introduced into the SRP RNA at predetermined positions will be used to identify neighborhoods at the single nucleotide and amino acid level. (3) Biophysical characterization of an RNA tetranucleotide loop interaction with protein SRP19. These studies will focus on the specific features of a critical protein-RNA interaction in a conserved region of the SRP and define its relationship to the overall process of protein translocation.
| Rosenblad, Magnus Alm; Larsen, Niels; Samuelsson, Tore et al. (2009) Kinship in the SRP RNA family. RNA Biol 6:508-16 |
| Iakhiaeva, Elena; Hinck, Cynthia S; Hinck, Andrew P et al. (2009) Characterization of the SRP68/72 interface of human signal recognition particle by systematic site-directed mutagenesis. Protein Sci 18:2183-95 |
| Ilangovan, Udayar; Bhuiyan, Shakhawat H; Hinck, Cynthia S et al. (2008) A. fulgidus SRP54 M-domain. J Biomol NMR 41:241-8 |
| Wower, Jacek; Wower, Iwona K; Zwieb, Christian (2008) Making the jump: new insights into the mechanism of trans-translation. J Biol 7:17 |
| Wang, Renjing; Ilangovan, Udayar; Robinson, Angela K et al. (2008) Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain. Biochemistry 47:8007-15 |
| Iakhiaeva, Elena; Wower, Jacek; Wower, Iwona K et al. (2008) The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72. RNA 14:1143-53 |
| Loverde, Philip T; Osman, Ahmed; Hinck, Andrew (2007) Schistosoma mansoni: TGF-beta signaling pathways. Exp Parasitol 117:304-17 |
| Yin, Jiaming; Iakhiaeva, Elena; Menichelli, Elena et al. (2007) Identification of the RNA binding regions of SRP68/72 and SRP72 by systematic mutagenesis of human SRP RNA. RNA Biol 4:154-9 |
| Iakhiaeva, Elena; Bhuiyan, Shakhawat Hossain; Yin, Jiaming et al. (2006) Protein SRP68 of human signal recognition particle: identification of the RNA and SRP72 binding domains. Protein Sci 15:1290-302 |
| Andersen, Ebbe Sloth; Rosenblad, Magnus Alm; Larsen, Niels et al. (2006) The tmRDB and SRPDB resources. Nucleic Acids Res 34:D163-8 |
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