title: Kinetic analysis of interaction of eukaryotic release factor 3 with guanine nucleotides.
abstract: Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3.mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K(d), approximately 70 microM) is about 70-fold lower than for GDP (K(d), approximately 1 microM) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP) approximately 2.4 s(-1); k(-2)(mant-GTP) approximately 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg(2+) concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K(d) approximately 0.7 microM) due to lowering of the dissociation rate constant approximately 24-fold (k(-1)(mant-GTP) approximately 0.14s(-1) approximately 0.14 s(-1)). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1 x eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.
authors.affiliation: Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York 11203, USA.
authors.forename: Vera P
authors.initials: VP
authors.lastname: Pisareva
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Pisarev
authors.forename: Christopher U T
authors.initials: CU
authors.lastname: Hellen
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Pestova
mesh_terms: D000818:Animals; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013816:Thermodynamics; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010454:Peptide Termination Factors; D062367:ortho-Aminobenzoates; C087971:peptide-chain-release factor 3; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate