Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.

The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.