Elongation Factors EF Tu and EF G Interact at Related Sites on Ribosomes

Abstract

The binding of elongation factor EF G to ribosomes inhibits the subsequent reaction of the ribosomes with the ternary complex aminoacyl-tRNA EF Tu GTP. Both the hydrolysis of GTP and the binding of aminoacyl-tRNA to ribosomes are nearly abolished by the previous binding of factor EF G to ribosomes in the presence of either fusidic acid plus either GTP or a nonhydrolyzable analog of GTP. The results suggest that each elongation factor binds to the same region on the ribosome. The GTPase activities of both factors EF G and EF Tu may be activated by interaction at the same ribosomal site, as has been previously suggested by others.In the process of protein biosynthesis, the hydrolysis of GTP is required for the addition of each succeeding amino acid to a growing peptide (for a recent review, see ref 1). During the elongation stage, GTP participates with elongation factor EF Tu in the binding of aminoacyl-tRNA to the ribosome as part of the intermediate aminoacyl-tRNA*EF Tu GTP complex (2, 3). When this complex interacts with the ribosome, GTP is hydrolyzed, aminoacyl-tRNA is bound to the ribosome, and EF Tu GDP is released (4, 5). After peptide bond formation, hydrolysis of GTP, catalyzed by factor EF G, is required for ejection of the deacylated tRNA (6) and for the translocation of the ribosome along the message (7, 8). Thus, two molecules of GTP appear to be required for the addition of each amino acid.The role of GTP hydrolysis in these reactions remains unclear. Among many unsolved questions, it is not known whether there are one or several GTPase enzymes on the ribosome, or whether the elongation factors are themselves GTPases. Recent reports that treatment of ribosomes with the antibiotic thiostrepton inhibits both EF G-and EF Tcatalyzed GTP hydrolysis (9-11) support the proposal that both factors interact at the same site on the ribosome. If we consider that the substrates for the site, aminoacyl-tRNA-EF Tu-GTP and EF G + GTP, appear to be widely different in structure and function, this is a surprising conclusion. Since it is possible that thiostrepton might inhibit two independent sites, a direct test was made to see whether one of the factors inhibited the activity of the other.To this end, experiments were undertaken to see whether binding factor EF G to the ribosome inhibited the hydrolysis of GTP in Phe-tRNA EF Tu GTP (referred to here as the "ternary complex"). These studies show that factor EF GI when bound to ribosomes in the presence of either the nonhydrolyzable GTP analogue guanylyl-5'-methylenediphosphonate (GMPPCP) (12), or fusidic acid plus GTP (13)

Identity of the Ribosomal Proteins Involved in the Interaction with Elongation Factor G

“…(i) Thiostrepton, an antibiotic that binds to 50S subunits, prevents both the EF-Gand EF-Tu-associated hydrolyses (30). (ii) Protein L7 and/or L12 are required for both reactions (15,16 hydrolytic reactions cannot occur simultaneously on a single ribosome (2,(11)(12)(13). Additionally, it has recently been suggested that GTP hydrolysis during initiation might also involve this ribosomal site (31).…”

“…An experimental approach to this problem is provided by the "single step" hydrolysis of GTP by EF-G and the 50S ribosomal subunit in the presence of fusidic acid (9,10). This system is one of the simplest manifestations of GTP participation in protein synthesis, and recent results suggest that it may involve a ribosomal site in common with the other GTP-requiring elongation step of protein synthesis, aminoacyl-tRNA binding (2,(11)(12)(13)(14).…”

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

“…Translocation of the mRNA:tRNA complex is promoted by the GTPase EF-G in order to vacate the A site so that the next incoming aminoacyl tRNA can be accommodated. Although there is considerable evidence indicating that the elongation factors (and other GTPases involved in the translation cycle) bind to overlapping sites on the ribosome (Cabrer et al 1972;Miller 1972;Richter et al 1972;Moazed et al 1988;Cameron et al 2002;Frank 2003), there must be structural distinctions between the different functional states of the ribosome that allow for their specificity of action (Richman and Bodley 1972;Wool et al 1992;Zavialov and Ehrenberg 2003). Traditionally, pre-translocationstate ribosomes have been characterized by the lack of reactivity of bound peptidyl tRNA substrate with incoming aminoacyl tRNA substrates.…”

The Interaction of Elongation Factor G with N -Acetylphenylalanyl Transfer RNA·Ribosme Complexes

“…In recent work, these reactions have been used to define the site of action of EF-G on the ribosomal surface (the G site). It appears that the G and A sites overlap on the ribosomal surface, for EF-G and EF-Tu cannot interact simultaneously with the ribosome (11)(12)(13)(14)(15)(16). If so, they must probably alternate in their binding to lpolysomal ribosomes during peptide chain elongation.…”