Effect of polaymines on yeast cell-free protein synthesizing system. I. Influence of spermine and spermidine on aminoacyl-tRNA transfer reaction.

Spermine and spermidine added to a Saccharomyces cerevisiae cell-free protein synthesizing system increased phenylalanine polymerization reaction several-fold at suboptimal concentration of Mg2+ and approximately two-fold at optimal amounts of Mg2+. The addition of polyamines greatly stimulated the enzymatic and nonenzymatic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to ribosomes. The binding of the acetylated derivative was higher than phenylalanyl-tRNA, however, as it was shown the former was bound exclusively to the A site of the ribosome. Contrary to the binding process, the puromycin reaction was not stimulated by spermine added at a concentration which enhanced the polyphenylalanine synthesis. These results indicate that polyamines have not only a sparing effect on the Mg2+ requirement for yeast protein synthesis in vitro and suggest that one of the possible sites of polyamines action might be the binding of aminoacyl-tRNA to ribosomes.     

Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids.

Homogenates of rat liver obtained 3 or 14 days after partial hepatectomy were used to prepare the postmicrosomal pH5-supernatant fraction and to prepare salt-wash fractions of the 40S ribosomal subunits and the 80S ribosomes. The factor-dependent binding of methionyl-tRNAfMet to ribosomes and the elongation-factor-1-dependent binding of phenylalanyl-tRNA to ribosomes were both increased after 3 days of growth, but not after 14 days of growth. An activity inhibitory to phenylalanyl-tRNA binding that was located in ribosomal wash fractions was decreased after 14 days of growth. Since the decreased inhibitory activity was obtained from the ribosomes and was tested against ribosomes and excess of pH5-supernatant fraction from control rat liver, its action was separate from the phenylalanyl-tRNA binding activities of the pH5-supernatant fractions from sham-operated and regenerating liver.     

Effect of modeccin on the steps of peptide-chain elongation.

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.     

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes

Conformational change of 50S ribosomes takes place during protein synthesis. The primary change is most likely in the secondary or tertiary structure of rRNA in the L7/L12 stalk region. In order to throw further light on this conformational change, the change in fluorescence of tight couple 50S ribosomes on conversion to loose couple 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-l-sulphonic acid-labelled L7/L12, following the treatment with elongation factor-G and 5′-guanylyl methylene diphosphate was measured. It was enhanced in agreement with the results reported earlier. Further, the quenching of fluorescence of 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-1-sulphonic acid-labelled L7/L12 by acrylamide was studied. The quenching is more in case of loose couples. On conversion of loose couple 50S ribosomes to tight couple ones the quenching becomes less whereas the reverse happens on conversion of tight couple 70S ribosomes to loose couples. These results indicate the conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.     

Conformational change of 23S RNA in 50S ribosome is responsible for translocation in protein synthesis

Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor -G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.     

IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bond formation

The aim of the present study was to investigate homoharringtonine alkaloid effect on: (i) the nonenzymatic and eEF-1-dependent Phe-tRNAPhe binding to poly(U)-programmed human placenta 80 S ribosomes; (ii) diphenylalanine synthesis accompanying nonenzymatic Phe-tRNAPhe binding; and (iii) acetylphenylalanyl-puromycin formation. Neither nonenzymatic nor eEF-1-dependent Phe-tRNAPhe binding were noticeably affected by the alkaloid, whereas diphenylalanine synthesis and puromycin reaction were strongly inhibited by homoharringtonine. It has been proposed that the site of homoharringtonine binding on 80 S ribosomes should overlap or coincide with the acceptor site of the ribosome.     

The action of virginiamycin M on the acceptor, donor, and catalytic sites of peptidyltransferase

Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.     

Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.     

Aminoacyltransferase I-catalysed binding of phenylalanyl-transfer ribonucleic acid to muscle ribosomes from normal and diabetic rats

The aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA (unfractionated Escherichia coli B tRNA acylated with radioactive phenylalanine and 19 non-radioactive amino acids) to skeletal-muscle ribosomes from diabetic rats was less than that to ribosomes from normal rats when the Mg(2+) concentration was low (7.5mm); whereas just the reverse was true when the concentration of the cation was higher (15mm). Thus the Mg(2+) dependency of aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA to ribosomes from normal and diabetic rats paralleled the effect of Mg(2+) concentration on synthesis of polyphenylalanine reported before. During incubation at 7.5mm-Mg(2+) phenylalanyl-tRNA was bound only to ribosomes bearing nascent peptidyl-tRNA. There are fewer such ribosomes in a preparation from the muscle of diabetic animals because diabetic animals synthesize less protein in vivo. Thus the difference in polyphenylalanine synthesis in vitro is adequately explained by the difference in enzyme-catalysed binding of phenylalanyl-tRNA to ribosomes, however, the basis of the difference in protein synthesis in vivo is still unknown.     

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.     

Pressure-induced dissociation of tight couple ribosomes

Ribosomes from Escherichia coli have been shown to undergo subunit dissociation at elevated hydrostatic pressure. This holds for both crude and highly purified ribosomes. No inhibitory effect could be detected by addition of either the S100 supernatant, or tRNA, polyuridylic acid, and spermine. Light scattering experiments at pressures up to 1000 bar reveal different susceptibility of tight couple and loose couple ribosomes toward pressure dissociation. Tight couples are subjected to EF-Tu-catalyzed binding of aminoacyl-tRNA, thus yielding a model system of the elongating ribosome before the peptidyl transfer step. High pressure dissociation of this compound suggests that enzymatic binding converts tight couples into loose couples. A hypothesis referring to conformational changes during the elongation cycle is presented.     

Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg²⁺ concentrations above 4 mm is a stringent test for activity.Fresh extracts, prepared at 10 mm-Mg²⁺ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles.In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg²⁺ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating.The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.     

Function of ribosomes and glutamyl-tRNA isoacceptors in protein synthesis in regenerating skeletal muscle

Ribosomes from 8-day-regenerating rat skeletal muscle have been shown to be more active in poly(U)-directed polyphenylalanine synthesis than ribosomes from control muscle. This difference persists after salt washing of the ribosomes and does not appear to be due to the presence of ribonuclease associated with the control ribosome population. Ribosomes from control muscle were also less active than those from regenerates in the nonenzymatic binding of phenylalanyl-tRNA to ribosomes and in the peptidyltransferase reaction. Three glutamyl-tRNA isoacceptors have been isolated from 8-day-regenerating rat skeletal muscle by preparative RPC-5 chromatography of total tRNA charged with [3H]glutamic acid. The two major isoacceptors observed, tRNAgluI and tRNAgluIII, respond to the glutamic acid codons GAG and GAA, respectively. A third, minor glutamyl isoacceptor, tRNAgluII, also responds to the codon GAA. When the three isoacceptors were tested for function in a polysomal cell-free protein synthesizing system, it was found that their relative levels of utilization were essentially identical to their relative abundances. Thus, the tRNA which increases in relative amount after the induction of regeneration, tRNAgluII, is not preferentially utilized for overall muscle protein synthesis.     

Properties of elongation factor G: its interaction with the ribosomal peptidyl-site

EF-G bound to poly(U)·ribosomes prevents enzymatic or nonenzymatic binding of charged tRNA not only to the A-site but also to the P-site. In turn, charged tRNA bound either to the P- or A-site prevents formation of EF-G·GMPPCP·ribosome complex. Ribosomes carrying newly synthetized peptidyl-tRNA in pretranslocative state are also unable to form stable complexes with EF-G. The functional implications of these observations are discussed and it is suggested that tRNA plays a regulatory role in the interaction of EF-G with ribosomes during the cyclic process of elongation.