Reconstitution of a GTPase Activity a 50S Ribosomal Protein from <Emphasis Type="Italic">E. coli</Emphasis>

DURING each step of peptide chain elongation the ribosome shifts up one triplet along the messenger RNA with concomitant movement of the peptidyl-transfer RNA from the donor to the acceptor site. This process, commonly known as translocation, is triggered by a supernatant protein, factor G, which in association with the ribosome cleaves GTP into GDP and inorganic phosphate^1,2 and it has been argued that the energy liberated in this reaction is used “to carry the complex one triplet forward”³.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.     

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.     

Stimulation of peptide elongation by thyroxine.

This study suggests that thyroxine stimulates peptide elongation in a cell-free rat liver polyribosome system. The thyroxine effect persists in the presence of sufficient aurintricarboxylic acid to prevent polyuridylic acid-stimulated peptide initiation. In addition, thyroxine stimulates elongation of pre-existing polyphenylalanine chains providing conclusive evidence that the effect does not depend on peptide initiation. Thyroxine does not stimulate release of nascent peptides from ribosomes into the supernatant phase of the reaction mixture. Therefore in this protein-synthesis system the thyroxine effect is expected to occur at one or more of the reactions of peptide chain elongation, which include aminoacyl-tRNA binding, peptide bond synthesis and translocation.     

Requirements for the initiation of polyphenylalanine synthesis by recombined ribosomal subunits from yeast

A reaction stimulated by elongation factor 2 (EF-2) is necessary for the formation of the initial peptide-bond in poly(U)-directed peptide synthesis on yeast ribosomal subunits. The stimulation is inhibited by fusidic acid or diphtheria toxin together with NAD. It is assumed that the reaction of EF-2 represents translocation: in the presence of EF-2 almost twice the amount of N-acetyl-phenylalanyl-tRNA is bound to ribosomes and moreover, upon addition of puromycin a considerable stimulation of N-acetyl-phenylalanyl-puromycin formation is found. Contradictory data in the literature on this subject may be due to nonenzymatic translocation caused by a high monovalent cation concentration in the ribosome preparations or to an unspecific method for the extraction of N-acetyl-phenylalanyl-puromycin.     

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 Å away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S subunit before its first round of translation.     

Amino acid incorporation in a cell-free system derived from rat liver studied with the aid of selenodiglutathione

Selenodiglutathione (GSSeSG), a potent inhibitor of elongation factor 2 (EF2) has been used to study amino acid incorporation in a rat liver cell-free system. While translocation of the ribosomes was inhibited by GSSeSG, ribosomes with a free acceptor site were still capable of incorporating one amino acid residue. From this the average number of amino acids incorporated per ribosomes was calculated to be 2--5. In this respect virtually no difference has been observed between ribosomes present on small or large aggregates. The time required for one translocation by all active ribosomes, and the time required for the incorporation of one amino acid (starting with aminoacyl-tRNA or amino acids) has also been determined. By incubation under conditions for amino acid incorporation, part of the ribosomes were completely inactivated whereas the rest remained as active as at the start of the incubation.     

Polypeptide Formation and Polyribosomes in Escherichia coli Treated with Chloramphenicol

In Escherichia coli cultures maximally inhibited with chloramphenicol, formation of polypeptides still continued at a slow, constant rate for at least 90 min. The rate of leucine incorporation was reduced to 0.5%, but methionine was only reduced to 2%, suggesting that chains are normally initiated with methionine but are prematurely released at a short chain length. Consistent with this possibility was the distribution of the products on Sephadex columns: a range of peptides longer than 4 and shorter than 60 to 70 residues was seen. Less than 10% of the peptides broke down during a chase with cold amino acids, and during continuous labeling they accumulated progressively. On the average, one peptide was formed per ribosome every 5 min. Peptide synthesis in the presence of chloramphenicol was still dependent on ribosome translocation; it stopped in a mutant with an inactivated temperature-sensitive elongation factor G. But even in the absence of translocation, new messenger ribonucleic acid (mRNA) chains were found joined to one or a few ribosomes. The chains had a size distribution comparable to that of mRNA from polyribosomes of growing cells. They were stabilized for an average time of about 5 min, but were more rapidly degraded after puromycin was added to the cells. This suggests that stabilization may be related to the average time spent by a ribosome on an mRNA chain, with or without polypeptide formation.     

A novel reaction of reticulocyte peptide-chain elongation factor, EF2, with guanosine nucleotides

The formation of phenylalanyl puromycin from phenylalanyl-tRNA, bound nonenzymically or enzymically to reticulocyte ribosomes, requires the peptide-chain elongation factor, EF2², and GTP. However the GTP analogue, GDPCP, may replace GTP to a significant extent in this reaction. Other purine or pyrimidine nucleotides have little or no activity. Multistep experiments with either GTP or GDPCP indicate that binding of EF2 to the ribosome for subsequent peptide formation may be a portion of the activity of the EF2 (independent of the translocation reaction) during the elongation process. Neomycin inhibits the formation of phenylalanyl puromycin using either GTP or GDPCP in this system.     

Factors involved in increased protein synthesis in rat kidney after administration of DDT

Homogenates of kidney cortex obtained from control rats and rats treated with DDT have been separated into microsomes or ribosomes, and into postmicrosomal (S105) supernatant fraction or pH 5 supernatant fraction. The incorporation of [¹⁴C]phenylalanyl-tRNA into peptide was increased when microsomes derived from kidneys of DDT-treated rats were incubated with pH 5 supernatant fraction from control rats. Elongation factors (EF) 1 and 2, necessary for the binding of aminoacyltRNA to ribosomes and for translocation of peptidyl-tRNA from the A site to the P site of ribosomes, were present in the pH 5 supernatant fractions of kidney of control and DDT-treated rats and these fractions were incubated with KCl-washed ribosomes obtained from livers of control rats. The results provided evidence that the increased incorporation observed with the pH 5 supernatant fraction obtained from the DDT-treated animals could not be attributed to decreased ribonuclease activity or to increased elongation factor 2 activity but was due to an increase in elongation factor l activity.     

Inhibition of the aminoacylation of selected tRNA molecules by an estrogen-regulated factor on uterine ribosomes. Regulation of aminoacylation of tRNA by estrogens

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen-deprived rats. The extracted inhibitor or a native inhibitor-ribosome complex affects the rate of the peptide elongation reaction in a uterine cell-free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl-tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro greater than Val greater than Arg greater than Try greater than Leu greater than Glu greater than Ile greater than Gly greater than His greater than Ser greater than Lys. Inhibition results from a specific dose-dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl-tRNA synthetase. The effect does not result from removal of A-C-C terminal nucleotides from the 3' end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl-tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol-induced inactivation of a ribosome-associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl-tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl-tRNAs to become rate-limiting in the sequential elongation of peptides.     

Elongation factors in protein biosynthesis

Translation elongation factors are the workhorses of protein synthesis on the ribosome. They assist in elongating the nascent polypeptide chain by one amino acid at a time. The general biochemical outline of the translation elongation cycle is well preserved in all biological kingdoms. Recently, there has been structural insight into the effects of antibiotics on elongation. These structures provide a scaffold for understanding the biological function of elongation factors before high-resolution structures of such factors in complex with ribosomes are obtained. Very recent structures of the yeast translocation factor and its complex with the antifungal drug sordarin reveal an unexpected conformational flexibility that might be crucial to the mechanism of translocation.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation.

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.     

Ribosomal translocation assayed by the matrix-bound poly(uridylic acid) column technique.

The system of translation of cellulose-bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre-translocation state ribosomes in columns. Translocation has been induced by passing the elongation factor G (EF-G) with GTP or its non-cleavable analog (guanosine 5'-[beta, gamma-methylene]triphosphate) through the column. A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation-inducing factors, has been proposed. The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column. The determination of the rate and amount of translocation has been done under different ionic conditions. It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF-G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF-G to become effective. Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF-G.     

Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.     

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.