Ribosomal translocation promoted by guanylylimido diphosphate and guanylyl‐methylene diphosphonate

Modolell, Juan; Girbés, Tomás; Vázquez, David

doi:10.1016/0014-5793(75)80429-7

Cited by 38 publications

(10 citation statements)

References 11 publications

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“…Moreover, our peptidyl transferase activity measurements, rRNA-directed chemical probing, and mRNA toeprinting show that EF-G catalyzes translocation of a pre-translocation complex exclusively in its GTP form, while EF-GÁGDPÁfusidic acid, which is able to stabilize the hybrid state, does not support completion of translocation. Our findings are in good agreement with most previous reports (Inoue-Yokosawa et al 1974;Belitsina et al 1975Belitsina et al , 1979Modolell et al 1975;Girbes et al 1976). How does EF-G catalyze the second step of translocation?…”

Section: Discussionsupporting

confidence: 94%

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Spiegel

Ermolenko²,

Noller³

2007

RNA

123

135

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Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDPÁfusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyltRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-GÁGDP. Unexpectedly, translocation with EF-GÁGTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-GÁGDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDPÁfusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-GÁGTP or EF-GÁGDPNP.

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Section: Discussionsupporting

confidence: 94%

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Spiegel

Ermolenko²,

Noller³

2007

RNA

123

135

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“…Our results (Table 3) indicate that GMP-P(NH)P is a much better substitute for GTP than GMP-P(CH2)P in ternary complex formation. A similar observation has been made by Kaziro's group in their study of peptidyl-tRNA translocation dependent on E. coli elongation factor G and GTP (Kaziro, Y., personal communication ; also see [25]). We have observed that GDP is a specific inhibitor of ternary complex formation promoted by A .…”

Section: Discussionsupporting

confidence: 75%

Role of GTP in Eukaryotic Polypeptide‐Chain Initiation

Ochiai-Yanagi

Mazumder

1976

European Journal of Biochemistry

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1.A factor, which makes a ternary complex with GTP and eukaryotic initiator tRNA (MettRNAi), has been purified 100-fold from developed cysts of Artemia salina. Some of the properties of the purified factor have been studied.2. M 2 + appears to inhibit ternary complex formation. 3. Little or no ternary complex is formed when 5 pM GTP is replaced by an identical concentration of UTP, CTP or ATP. The analog, guanosine 5'-(fi,y-imino)triphosphate [GMP-P(NH)P] seems to be a much better substitute for GTP than guanosine 5'-(B~-methylene)triphosphate [GMP-P(CH2)PI. Since GMP-P(NH)P is as effective as GTP in ternary complex formation, it would appear that GTP plays the role of an allosteric effector in this step of eukaryotic polypeptide chain initiation.4. GDP inhibits both the rate and extent of ternary complex formation. The inhibition is largely reversed by adding a 5-fold molar excess of GTP over GDP. dGDP is slightly less inhibitory than GDP. UDP and CDP are much less inhibitory than GDP and very little inhibition is obtained with ADP.5. The preformed ternary complex is rapidly and completely destroyed in the presence of N-ethylmaleimide. The results suggest that free -SH groups of the factor may be essential for maintaining the integrity of the ternary complex.Despite much work on the mechanism of polypeptide chain initiation in eukaryotic cells, relatively little information is presently available regarding the precise role of GTP in this process. For example, our understanding of the step(s) in which GTP plays an allosteric role, of the step(s) in which GTP is hydrolyzed and of the role of GTP hydrolysis in eukaryotic polypeptide chain initiation, is either incomplete or lacking. In order to clarify the above problems, we have recently undertaken a project directed toward the purification and detailed examination of the properties of eukaryotic protein factor(s) whose functioning in the process of polypeptide chain initiation is either dependent on and/or modified by guanine nucleotides. In the present paper, a procedure is described for 100-fold purification of such a factor from developed cysts of the brine shrimp, Artemiu Ahhrrviations. GMP-P(CH2)P and GMP-P(NH)P, guanosine S'-(p,.;-methylene)triphosphate and guanosine S'-(p,y-imino)triphosphate, respectively; IF-2, prokaryotic initiation factor 2; MalNEt, N-ethylmaleimide, -~ ~ salina. The purified factor forms a ternary complex with GTP and initiator Met-tRNA (Met-tRNAi) of eukaryotic origin. The role of GTP in this reaction appears to be that of an allosteric effector since the analog GMP-P(NH)P is as effective as GTP in ternary complex formation. GMP-P(CH2)P seems to be much less active than GMP-P(NH)P. Although GDP inhibits ternary complex formation, the inhibition is, reversed by increasing concentrations of GTP. We have also observed that the preformed factor . GTP . MettRNAi ternary complex is rapidly destroyed in the presence of N-ethylmaleimide. These and other properties of the purified faFtor are described in this paper.Previous work from other l...

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“…1). In contrast, the three nucleotides have wholly different activities in translocation of peptidyltRNA: GTP is at least 100 times more effective than the analogs and competes very efficiently with them [31]. Translocation is clearly much more selective in terms of nucleotide requirements than the mere binding of EF-G to the ribosome.…”

Section: Discussionmentioning

confidence: 99%

The Formation of Guanosine-Nucleotide . Elongation-Factor-G . Ribosome Complexes on Free 70-S Ribosomes, 50-S Subunits, and Polysomes. A Comparative Study

San‐Millán¹,

Vázquez²,

Modolell³

1977

Eur J Biochem

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The ability of free 70‐S ribosomes, 50‐S subunits and purified, endogenous Escherichia coli polysomes to form complexes with elongation factor G (EF‐G) and different concentrations of guanosine nucleotides has been examined. GTP (plus fusidic acid) has been used with all these types of ribosomal particles and GDP (plus the antibiotic), guanyl‐5′‐yl methylenediphosphonate (Guo‐5′‐P2‐CH2‐P) and guanyl‐5′‐yl imidodiphosphate (Guo‐5′‐P2‐NH‐P) with free 70‐S ribosomes. In every case the data, obtained with the nitrocellulose membrane filtration technique and under saturating concentrations of EF‐G and fusidic acid (when present), fell on one straight line in Scatchard plots, thus indicating that the sites involved in guanosine nucleotide binding are homogeneous and lack cooperative interactions. With 70‐S ribosomes between 0.6 and 0.75 molecule of nucleotide were bound per ribosome at saturating concentrations of guanosine nucleotide (3–10 μM). Assuming that about one‐third of ribosomes in the preparations were inactive, this result suggests that with each of the four guanosine nucleotides one molecule of this ligand was bound per ribosome. The apparent affinity constants Ka were: for GTP = 7.7 × 106 M−1, for GDP = 1.3 × 107 M−1, for Guo‐5′‐P2‐CH2‐P= 5.0 × 106 M−1 and for Guo‐5′‐P2‐NH‐P= 1.9 × 106 M−1. 50‐S subunits, under the conditions used (10 mM Mg2+ and 65 mM NH+4) and at saturating concentrations of GTP, only complexed about 0.35 molecule of nucleotide per subunit and the apparent KGTPa was 1.2 × 106 M−1. Addition of 30‐S subunits to the 50‐S subunits raised the number of molecules complexed by 50‐S particles to 0.5 and increased KGTPa to 5.1 × 106 M−1, suggesting an important role of the 30‐S subunit in the configuration of the nucleotide binding site. With purified endogenous polysomes, washed with either 1 M or 0.1 M NH4Cl, only 0.2–0.25 molecule of GTP was bound per ribosome at saturation and KGTPa was 6.7 × 106 M−1. Deacylation of nascent peptidyl chains raised these values to about 0.5 and 9.6 × 106 M−1, respectively. The small fraction of ribosomes that formed the complex in untreated polysomes appeared to be constituted mainly of true polysomal ribosomes, since (a) they possessed peptidyl‐tRNA, as judged by the increased stability of their GDP · EF‐G · ribosome · fusidic‐acid complex in the presence of puromycin, and (b) they sedimented as polysomes. The results thus indicate the existence of a functional heterogeneity in the polysomal preparations.

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Ribosomal translocation promoted by guanylylimido diphosphate and guanylyl‐methylene diphosphonate

Cited by 38 publications

References 11 publications

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Role of GTP in Eukaryotic Polypeptide‐Chain Initiation

The Formation of Guanosine-Nucleotide . Elongation-Factor-G . Ribosome Complexes on Free 70-S Ribosomes, 50-S Subunits, and Polysomes. A Comparative Study

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