INTERACTION OF GUANINE-NUCLEOTIDES WITH THE SIGNAL RECOGNITION PARTICLE FROM ESCHERICHIA-COLI

The bacterial signal recognition particle (SRP) is an RNA-protein comp lex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5 S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membra ne targeting of translating ribosomes. In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding t o Ffh in different functional complexes. The association and dissociat ion rate constants of GTP/GDP binding to Ffh were measured using a flu orescent analogue of GTP/GDP, mant-GTP/GDP. For both nucleotides, asso ciation and dissociation rate constants were about 10(6) M-1 s(-1) and 10 s(-1) respectively. The equilibrium constants of nonmodified GTP a nd GDP binding to Ffh alone and in SRP, and in the complex with the ri bosomes were measured by competition with mant-GDP. In all cases, the same 1-2 mu M affinity for GTP and GDP was observed. Binding of both G TP and GDP to Ffh was independent of Mg2+ ions. The data suggest that, at conditions in vivo, (i) there will be rapid spontaneous GDP-GTP ex change, and (ii) the GTP-bound form of Ffh, or of SRP, will be predomi nant.