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The interaction of signal sequences with the signal recognition particle
The signal recognition particle (SRP) is an RNA-protein complex that directs proteins containing an N-terminal signal sequence into the secretory pathway. For high fidelity in the selection of substrates for secretion, the SRP must recognize the features of signal sequences that uniquely distinguish them from other segments of proteins. The recognition of signal sequences by SRP is therefore almost certain to involve novel modes of protein-peptide interaction. Based on crosslinking data and the crystal structure of the SRP protein subunit Ffh, it is widely assumed, though unsubstantiated by experimental data, that the signal sequence binds to a hydrophobic groove on the “M-domain” of Ffh. However, in this thesis data are presented from crosslinking experiments and from direct binding assays that strongly suggest the adjacent “NG-domain” of Ffh contributes a substantial portion of the binding site for signal sequences. Using a zero-length crosslinking method previously untested in the SRP system, the binding site has been localized to the “G-domain”, a subdomain within the NG domain that has a tertiary fold which is to a large extent typical of ras-like GTPases. Given that previous crosslinking studies have suggested the M-domain is at least in close proximity to the signal peptide, it is likely the signal peptide binds close to the interface between the two domains. Defining how the two domains interact is therefore essential for understanding how the SRP recognizes signal sequences. How the domains interact was left uncertain by the extensive packing interactions between molecules in the crystal structure of Ffh. The work in this thesis has defined a segment of the M-domain, termed the “finger loop”, which interacts with the NG domain. Finally, this thesis work has also explored the functional role of the RNA in the SRP. Data are presented leading to a model whereby the RNA stabilizes conformational states of Ffh in which the two domains are more loosely associated with each other. This may explain previous studies reports that RNA stimulates the association of Ffh with its receptor FtsY.
Cleverley, Robert Matthew, "The interaction of signal sequences with the signal recognition particle" (2002). Doctoral Dissertations Available from Proquest. AAI3068547.