Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli.
The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S 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 membrane targeting of translating ribosomes. In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding to Ffh in different functional complexes. The association and dissociation rate constants of GTP/GDP binding to Ffh were measured using a fluorescent analogue of GTP/GDP, mant-GTP/GDP. For both nucleotides, association and dissociation rate constants were about 10(6) M-1 s-1 and 10 s-1, respectively. The equilibrium constants of nonmodified GTP and GDP binding to Ffh alone and in SRP, and in the complex with the ribosomes were measured by competition with mant-GDP. In all cases, the same 1-2 microM affinity for GTP and GDP was observed. Binding of both GTP 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 exchange, and (ii) the GTP-bound form of Ffh, or of SRP, will be predominant.
Jagath
J R
JR
Institute of Molecular Biology, University of Witten/Herdecke, Germany.
Rodnina
M V
MV
Lentzen
G
G
Wintermeyer
W
W
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D006150:Guanine Nucleotides; D018271:Signal Recognition Particle; D062367:ortho-Aminobenzoates; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium