The aim is to understand signal recognition particle (SRP) dependent targeting of membrane and secretory proteins to the translocation machinery of the cell, at the level of molecular structure and chemical mechanism. We have prepared, and solved structures for, the 'N' and GTPase domains of the prokaryotic homologue of the 54 kD protein component (Ffh) of the SRP from T. aquaticus and the entire Ffh including the signal sequence and SRP RNA binding M domain. We seek to understand binding of signal sequences at the level of affinity, specificity and three-dimensional structure. Mutations of hydrophobic residues that line the signal-binding site will be correlated with signal sequence binding and Ffh structure. Crystal structures will elucidate the mechanism by which binding of the SRP to its membrane bound receptor (SR) is coupled to GTPase activation. Mutations made in the conserved interface between 'N' and GTPase domains will elucidate the mechanism of allosteric coupling of nucleotide binding to receptor binding in unidirectional targeting to the membrane. The role of RNA in SRP structure and function and in mediating translation arrest of ribosomal synthesis will be defined using crystallography and electron microscopy. Components of the membrane pore are to be prepared and characterized for structural approaches to translocation.
