Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes.

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.     

The function of the translating ribosome: allosteric three-site model of elongation.

During the last decade, a new model for the ribosomal elongation cycle has emerged. It is based on the finding that eubacterial ribosomes possess 3 tRNA binding sites. More recently, this has been confirmed for archaebacterial and eukaryotic ribosomes as well, and thus appears to be a universal feature of the protein synthetic machinery. Ribosomes from organisms of all 3 kingdoms harbor, in addition to the classical P and A sites, an E site (E for exit), into which deacylated tRNA is displaced during translocation, and from which it is expelled by the binding of an aminoacyl-tRNA to the A site at the beginning of the subsequent elongation round. The main features of the allosteric 3-site model of ribosomal elongation are the following: first, the third tRNA binding site is located 'upstream' adjacent to the P site with respect to the messenger, ie on the 5'-side of the P site. Second, during translocation, deacylated tRNA does not leave the ribosome from the P site, but co-translocates from the P site to the E site--when peptidyl-tRNA translocates from the A site to the P site. Third, deacylated tRNA is tightly bound to the E site in the post-translocational state, where it undergoes codon--anticodon interaction. Fourth, the elongating ribosome oscillates between 2 main conformations: (i), the pre-translocational conformer, where aminoacyl-tRNA (or peptidyl-tRNA) and peptidyl-tRNA (or deacylated tRNA) are firmly bound to the A and P sites, respectively; and (ii), the post-translocational conformer, where peptidyl-tRNA and deacylated tRNA are firmly bound to the P and E sites, respectively. The transition between the 2 states is regulated in an allosteric manner via negative cooperatively. It is modulated in a symmetrical fashion by the 2 elongation factors Tu and G. An elongating ribosome always maintains 2 high-affinity tRNA binding sites with 2 adjacent codon--anticodon interactions. The allosteric transition from the post- to the pre-translocational state is involved in the accuracy of aminoacyl-tRNA selection, and the maintenance of 2 codon--anticodon interactions helps to keep the messenger in frame during translation.     

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.     

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

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 GDP.fusidic 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 peptidyl-tRNA 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-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, 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 GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.     

Elongation factor 2 from Artemia salina embryos and its affinity for ribosomes

Crude extracts from Artemia salina undeveloped embryos do not contain detectable elongation-factor-2 (EF2) kinase and endogenous ADP-ribosylating activities. Accordingly, EF2 purified from this source is an enzyme relatively free from phosphorylated and ADP-ribosylated forms. Endogenous ADP-ribosyltransferase activity appears only after purification of EF2. The affinities of EF2 and of ADP-ribosyl-EF2 for ribosomes from A. salina undeveloped embryos have been calculated by measuring the ability of the factors to inhibit the N-glycosidase activity of ricin on ribosomes.     

Timing of GTP binding and hydrolysis by translation termination factor RF3

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.     

Studies on elongation factor II from calf brain

Elongation factor II (EF2) has been purified from calf brain, and its reactions with guanosine nucleotides and ribosomes have been studied. Its behavior is, in general, similar to that observed with EF2 from other eukaryote sources. Thus, in the presence of GTP or GDP, EF2 interacts with ribosomes to form a ribosome-EF2-GDP complex. Fusidic acid has little effect on the stability of this complex, which suggests that it is more stable than the corresponding complex from prokaryote systems. As assayed by a nitrocellulose filter technique, only GTP, GDP, dGTP and GDPCP are bound to ribosomes dependent on EF2. In the absence of ribosomes, an EF2-GTP or EF2-GDP complex can be detected. Fusidic acid at relatively high concentrations inhibits their formation, but diphtheria toxin in the presence of NAD does not. The EF2-GTP complex has been separated from unbound GTP by gel filtration, and the reactivity of the complex with ribosomes has been investigated. When EF2-GTP is incubated with ribosomes, GTP hydrolysis occurs, and evidence for a ribosome-EF2-GDP complex has been obtained. The results thus suggest that the EF2-GTP complex may be an intermediate in the binding of EF2 to ribosomes. Based on molecular sieve chromatography, it appears that the stability of these complexes is ribosome-EF2-GDP > EF2-GTP > EF2-GDP.     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non-hydrolysable GTP analogues.

Treatment of reticulocyte polysomes with elongation factor eEF-2 and GTP led to an increased sensitivity of peptidyl-tRNA for puromycin as a result of the translocation from the ribosomal A-site to the P-site. Upon addition of an excess of the non-hydrolysable GTP analogue, GuoPP[CH2]P, the puromycin sensitivity decreased rapidly. The decrease in sensitivity required high concentrations of eEF-2 with half maximal effect at an eEF-2 concentration of around 1 microM. The data suggest either that peptidyl-tRNA had re-translocated back to the A-site due to the higher affinity of eEF-2 for the pre-translocation than for the post-translocation ribosome, or that the eEF-2-GuoPP[CH2]P complex blocks the peptidyl-transferase activity.     

Studies on the binding of bacterial elongation factors EF Tu and EF G to ribosomes

When EF G² from Escherichia coli or Pseudomonas fluorescens is pre-bound to ribosomes in the presence of GMD, or GTP and fusidic acid, a differential effect is observed on the subsequent EF Tu-catalyzed binding of aminoacyl-tRNA to ribosomes. The EF G from E. coli nearly completely prevents the binding reaction, whereas the corresponding factor from P. fluorescens displays a significantly lower inhibitory effect. Both EF G factors form stable complexes with ribosomes and are equally efficient in the polymerization reaction. The difference in inhibitory properties between the two factors persists over a wide range of NH₄Cl concentration.     

Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.     

Available sulphydryl groups of mammalian ribosomes in different functional states

The numbers of sulphydryl groups on NH₄Cl-washed rat liver polyribosomes in different functional states were measured under carefully standardized conditions with ¹⁴C-labelled N-ethylmaleimide and ³⁵S-labelled 5,5-dithio-bis(2-nitrobenzoic acid). Ribosomes denatured with urea had 120 titratable sulphydryl groups, 60 on each subunit, whereas native ribosomes invariably showed fewer available sulphydryl groups. Ribosomes stripped of transfer RNA (S-type ribosomes) had 55 available sulphydryl groups. Ribosomes bearing the growing peptidyl-tRNA at the acceptor site had 41 sulphydryl groups available. If these A-type ribosomes were labelled with ¹⁴C-labelled N-ethylmaleimide and dissociated into subunits, 23 of the labelled sulphydryl groups were found on the 60 S subunit and 19 on the 40 S subunit. After translocation of the peptidyl-tRNA to the donor position on ribosomes (D ribosomes), the number of available sulphydryl groups increased to 72, of which 43 were on the 60 S subunit and 29 on the 40 S subunit. This demonstrates that both subunits participate in the change of peptidyl-tRNA from the A to D positions. When the D ribosomes were reacted with EF2 (elongation factor) and GTP, the available sulphydryl groups increased to 82; addition of EF2 alone or with GDP, GDPCP or ATP failed to cause this increase, which has accordingly been attributed to an energy-dependent conformational change in the ribosome.Ribosomes were reconstructed from subunits with poly(U) and Phe-tRNA. In the presence of poly(U) only, a ribosome with 55 available SH groups was formed, thus corresponding to the stripped ribosomes. When both poly(U) and Phe-tRNA were present, a ribosome was formed with 44 available sulphydryl groups, corresponding approximately to an A-type ribosome. Since no EF1 or GTP was used in reconstructing this ribosome, these data indicate that the conformation of A-type ribosomes is not dependent on EF1 or GTP, but is due to the presence of tRNA at the acceptor site.We therefore incline to the view that the observed changes in available SH groups reflect conformational changes, with an opening up of ribosome structure as it progresses from having the peptidyl-tRNA at the A position to the D position and then binds EF2 and GTP, followed by a restoration of the more compact from when the incoming aminoacyl-tRNA is then bound.     

Is the three-site model for the ribosomal elongation cycle sound?

The release of deacylated tRNA from the ribosome as a result of translocation has been studied. Translating ribosomes prepared with poly(U)-S-S-Sepharose columns have been used. It has been shown that deacylated tRNA released from the ribosomal P site as a result of translocation rebinds with the vacated A site. Consistent with the known properties of the A site of the ribosome, this interaction is reversible, Mg2+-dependent, codon-specific and is inhibited by the antibiotic tetracycline. It has been concluded that the proposed three-site model of the ribosomal elongation cycle (Rheinberger and Nierhaus (1983) Proc. Natl. Acad. Sci. USA 80, 4213-4217) is not sound: the experimentally observed 'retention' of the deacylated tRNA on the ribosome after translocation can be explained by a codon-dependent rebinding to the A site, rather than by its transition to the 'E site', i.e., in terms of the classical two-site model.     

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

Interaction of elongation factor 2 from wheat germ with guanosine nucleotides and ribosomes.

1. The amino acid composition of wheat germ EF2 differs to some extent from that of elongation factors from mammals and bacteria. 2. The purified wheat germ EF2, similarly as the factors from other sources, is active in the: EF1-dependent polymerization of phenylalanine; ribosome-dependent GTP hydrolysis; binding of guanosine nucleotides; and ADP-ribosylation in the presence of diphtheria toxin. Fusidic acid at a concentration of 1 mM inhibits all these EF2-dependent reactions. 3. Diphtheria toxin in the presence of NAD+ inhibits polymerization of phenylalanine but does not effect GTP binding to EF2. 4. Binding of GDP to wheat germ EF2 is inhibited by ribosomes. During interaction with ribosomes, GTP in EF2-GTP complex is rapidly hydrolysed to GDP. Both GTP and 5'-guanylmethylenediphosphonate competitively inhibit formation of the ribosome-EF2-GDP complex due to the replacement of GDP from the complex. The latter is stabilized by fusidic acid.     

Binding of GDP to a ribosomal protein after elongation factor-2 dependent GTP hydrolysis.

Incubation of 80S ribosomes with a substoichiometric amount of [alpha-32P]GTP and with eEF-2 resulted in the specific labeling of one ribosomal protein which migrated very close to the position of the acidic phosphoprotein P2 from the 60S subunit in two-dimensional isofocusing-SDS gel electrophoresis. Localization of protein P2 in this electrophoretic system was ascertained by correlation with its position in the standard two-dimensional acidic-SDS gel electrophoresis after its specific phosphorylation by casein kinase II. Labeling of the ribosomal protein was dependent on the presence of eEF-2, and could be attributed to [alpha-32P]GDP binding from the results of chase experiments and HPLC identification, this binding being very likely responsible for the slight shift in the electrophoretical position of the protein. Incubation of ribosomes with tRNA(Phe) in the absence of mRNA induced the release of the bound GDP.     

Release of ribosome-bound ribosome recycling factor by elongation factor G

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.     

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.     

Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.