The role of cholesteryl 14-methylhexadecanoate in the function of eukaryotic peptide elongation factor 1

The binding of [3H]cholesteryl 14-methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5'-[beta, gamma-methylene]-triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14-methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959-966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP-dependent autophosphorylation of the protein-synthesis factor. Cholesteryl 14-methylhexadecanoate was bound only by the beta subunit of the factor. Addition of the alpha subunit, which was inactive on its own, stimulated the binding of the ester to the beta subunit in a sigmoid dependence. The binding of the ester was significantly stimulated by aminoacyl-tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14-methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)-dependent binding of phenylalanyl-tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14-methylhexadecanoate. The ester thus interacts with the GTP-dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14-methylhexadecanoate.     

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.     

Cholesteryl esters are bound by a peptide-initiation and a peptide-elongation factor.

Significantly higher quantities of cholesteryl 14-methylhexadecanoate than of cholesteryl laurate and cholesteryl palmitate are bound by a homogeneous peptide-initiation factor and purified peptide-elongation factor 1. Cholesteryl 14-methylhexadecanoate may function as a specific allosteric modifier changing the conformation of protein synthesis factors and thus modulating the activity of their binding sites.     

Intimal smooth muscle cells up-regulate beta-very low density lipoprotein-mediated cholesterol accumulation by enhancing beta-very low density lipoprotein uptake and decreasing cholesterol efflux

To clarify the mechanism of smooth muscle cell (SMC)-derived foam cell formation, we investigated beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in vascular medial SMCs (M-SMCs) from normal rabbits compared with intimal SMCs (I-SMCs) from normal rabbits fed a high-cholesterol diet and LDL receptor-deficient rabbits. For both types of I-SMCs, uptake of [3H]cholesteryl oleate labeled beta-VLDL increased 1.6 times and release of [3H]cholesterol decreased 40% compared with M-SMCs. M-SMCs took up part of the beta-VLDL through the LDL receptor but I-SMCs did not. mRNAs for the VLDL receptor and the LDL receptor relative with 11 ligand binding repeats were expressed at similar levels in all SMCs. M-SMCs expressed more LDL receptor-related protein than I-SMCs. Ligand blotting analysis revealed greater 125I-beta-VLDL binding to a 700-kDa protein in I-SMCs compared with M-SMCs. I-SMCs had higher activities of acid cholesterol esterase and acyl-CoA:cholesterol acyltransferase, and lower activity of neutral cholesterol esterase than M-SMCs in both the absence and the presence of beta-VLDL. These results indicate that I-SMCs accumulate more cholesteryl ester than M-SMCs by taking up more beta-VLDL and by effluxing less cholesterol.     

Sequence analysis of the peptide-elongation factor EF-2 gene, downstream from those of ribosomal proteins H-S12 and H-S7, from the archaebacterial extreme halophile, Halobacterium halobium

The gene for the peptide-elongation factor 2 (EF-2) was cloned from the archaebacterial extreme halophile Halobacterium halobium and sequenced. The 1013 nucleotides upstream from this gene was two open reading frames similar to ribosomal proteins S12 and S7 from Escherichia coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF-2 and eubacterial EF-G. Sequence similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.     

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.     

Metabolic fate of pancreas-derived cholesterol esterase in intestine: an in vitro study using Caco-2 cells

Bile salt-stimulated cholesterol esterase is synthesized in the pancreatic acinar cells and is released into the intestinal lumen where it catalyzes cholesterol absorption. In the current study, Caco-2 cells were used as an in vitro model to study the interaction between the pancreatic cholesterol esterase with intestinal cells. Results showed that addition of increasing concentrations of cholesterol esterase in the incubation medium increased the uptake of micellar cholesteryl oleate by Caco-2 cells. The cholesterol esterase also increased the cellular uptake of the nonhydrolyzable cholesteryl linoleoyl ether. However, maximum uptake of the cholesteryl ether analog was 50% of that for cholesteryl oleate. The initial interaction of cholesterol esterase with Caco-2 cells was mediated by binding of the protein to a low affinity and high capacity binding site on the cell surface. Cholesterol esterase bound to the cell surface could be internalized via a monensin-sensitive mechanism. The cholesterol esterase taken up by the cells had a short residence time and was either degraded or was rapidly re-secreted from the cells. Chloroquine had no effect on the degradation or re-secretion of cholesterol esterase by Caco-2 cells, indicating that lysosomes were not involved with these processes. The cholesterol esterase taken up by the cells was not available to mediate further cholesterol uptake. These results indicated that the bile salt-stimulated cholesterol esterase secreted from pancreas could facilitate intestinal lipid absorption only transiently. The data suggest that the regulation of cholesterol esterase synthesis and secretion by the pancreas may be important for regulation of cholesterol absorption.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins

Human HDL subfractions (HDL2, HDL3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [14C]cholesterol and [3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A2 from Crotalus adamanteus in presence of albumin leading to a 72-82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [14C]cholesterol from HDL3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [3H]cholesteryl ether was 2-4-times less important. Uptake of HDL cholesterol tended to saturate at 150-200 micrograms/ml HDL protein. A prior phospholipase treatment of HDL3 stimulated by 2-5-fold the uptake of [3H]cholesteryl ether, whereas the transfer of free [14C]cholesterol was minimally increased. The uptake of 3H/14C-labelled sterols from HDL2 was 2-3-times higher than from HDL3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37 degrees C, the specific uptake and degradation of HDL3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL3 with phospholipase A2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (X5). The dissociation was still more pronounced with phospholipase-treated HDL3. Competition experiments showed that 12-times more unlabelled HDL3 were required to half reduce the uptake of HDL3 [3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL2 was quantitatively comparable to that from HDL3. (C) Binding of 125I-HDL subfractions was followed at 4 degrees C. A specific binding was observed for HDL2 and HDL3, although kinetic parameters were quite different (KD of 9 and 25 micrograms/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.     

Binding of protein synthesis initiation factor IF1 to 30 S ribosomal subunits: effects of other initiation factors and identification of proteins near the binding site.

The effects of other components of the initiation complex on Escherichia coli initiation factor IFI binding to 30 S ribosomal subunits were studied. Binding of [¹⁴C]IF1 in the absence of other initiation complex components was slight. Addition of either IF2 or IF3 stimulated binding to a variable extent. Maximum binding was observed when both IF2 and IF3 were present. Addition of GTP, fMet-tRNA, and phage R17 RNA caused little or no further stimulation of [¹⁴C]IF1 binding. A maximum of 0.5 molecule of [¹⁴C]IF1 bound per 30 S subunit in the presence of an excess of each of the three factors over 30 S subunits.Complexes of 30 S subunits, [¹⁴C]IF1, IF2, and IF3 were treated with the bifunctional protein cross-linking reagent dimethyl suberimidate in order to identify the ribosomal proteins near the binding site for IF1. Non-cross-linked [¹⁴C]IF1 was removed from the complexes by sedimentation through buffer containing a high salt concentration, and total protein was extracted from the pelleted particles. Approximately 12% of the [¹⁴C]IF1 was recovered in the pellet fraction. The mixture of cross-linked products was analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Autoradiography of the gel showed radioactive bands with molecular weights of 21,000, 25,000, and many greater than 120,000. The results indicate that [¹⁴C]IF1 was cross-linked directly to at least two ribosomal proteins. Analysis of the cross-linked mixture by radioimmunodiffusion with specific antisera prepared against each of the 30 S ribosomal proteins showed radioactivity in the precipitin bands formed with antisera against S12 and S19, and in lower yield with those against S1 and S13. Antiserum against IF2 also showed [¹⁴C]IF1 in the precipitin band. The results show that [¹⁴C]IF1 was present in covalently cross-linked complexes containing 30 S ribosomal proteins S1, S12, S13 and S19, and initiation factor IF2. The same ribosomal proteins have been implicated in the binding sites for IF2 and IF3. The results suggest that the three initiation factors bind to the 30 S subunit at the same or overlapping sites.     

Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNA(fMet) and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNA(Met) or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8-10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNA(fMet) is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNA(fMet) and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1-2mm). The ternary complex of the factor with methionyl-tRNA(fMet) and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNA(fMet) not required for translation.     

Stimulation, by two Escherichia coli supernatant proteins, of the initiation of polypeptide synthesis.

Two protein factors (A and B) have been partially purified from Escherichia coli supernatant which, in combination, are more effective than 0.5 M NH4Cl in stimulating ribosomes for AcPhe-tRNA and fMet-tRNA binding, for the puromycin reaction, and for incorporating acetylphenylalanine from AcPhe-tRNA into polypeptide. The factors appear to differ from the initiation factors, the elongation factor EF-T, and ribosomal proteins. Some uncertainty exists as to whether factor B is different from EF-G. To maximize the effect of the factors in initiator tRNA binding, we preincubated the ribosomes with the factors and carried out the binding assay for a short period at 15 degrees C. Maximal stimulation of binding occurred after about a 2-min preincubation at 37 degrees C. Longer preincubation times were required at 15 degrees C, and only slight stimulation was observed after preincubation at 0 degrees C. The extent of stimulation by the factors was not affected when the NH4Cl concentration was increased from 40 to 500 mM in the preincubation. The presence of both the 30S and 50S ribosomal subunits is required for the enhancement of AcPhe-tRNA binding. Polyphenylalanine synthesis carried out without AcPhe-tRNA is inhibited by the factors. It is suggested that the factors may act by inducing a structural rearrangement of the ribosomes.     

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.     

Functional sites of interaction between release factor RF1 and the ribosome

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.     

Role of ribosome degradation in the death of starved Escherichia coli cells.

In Escherichia coli cultures limited for phosphate, the number of ribosomal particles was reduced to a small percentage of its earlier peak value by the time the viable cell count began to drop; the 30S subunits decreased more than the 50S subunits. Moreover, the ribosomal activity was reduced even more: these cells no longer synthesized protein, and their extracts could not translate phage RNA unless ribosomes were added. The translation initiation factors also disappeared, suggesting that they become less stable when released from their normal attachment to 30S subunits. In contrast, elongation factors, aminoacyl-tRNA synthetases, and tRNA persisted. During further incubation, until viability was reduced to 10(-5), the ribosomal particles disappeared altogether, while tRNA continued to be preserved. These results suggest that an excessive loss of ribosomes (and of initiation factors) may be a major cause of cell death during prolonged phosphate starvation.     

Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization

The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis. The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells. About 90% of this activity is found in the postribosomal supernatant of the cell. Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A. The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems. In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold. The binding reaction shows broad monovalent and divalent cation optima. The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors. Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E. coli 30 S or wheat germ 40 S subunits.     

Characterization of the domains of E. coli initiation factor IF2 responsible for recognition of the ribosome.

We have studied the interactions between the ribosome and the domains of Escherichia coli translation initiation factor 2, using an in vitro ribosomal binding assay with wild-type forms, N- and C-terminal truncated forms of IF2 as well as isolated structural domains. A deletion mutant of the factor consisting of the two N-terminal domains of IF2, binds to both 30S and 50S ribosomal subunits as well as to 70S ribosomes. Furthermore, a truncated form of IF2, lacking the two N-terminal domains, binds to 30S ribosomal subunits in the presence of IF1. In addition, this N-terminal deletion mutant IF2 possess a low but significant affinity for the 70S ribosome which is increased by addition of IF1. The isolated C-terminal domain of IF2 has no intrinsic affinity for the ribosome nor does the deletion of this domain from IF2 affect the ribosomal binding capability of IF2. We conclude that the N-terminus of IF2 is required for optimal interaction of the factor with both 30S and 50S ribosomal subunits. A structural model for the interaction of IF2 with the ribosome is presented.     

The influence of estradiol on cholesterol processing by the corpus luteum

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.     

A model for the interaction of the G3‐subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet‐tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2‐30S interaction, is positioned between the GTP‐binding G2 and the fMet‐tRNA binding C‐terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β‐sheets with each four anti‐parallel strands, followed by a C‐terminal α‐helix. In line with its role as linker between G3 and subdomain C1, this helix has no well‐defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB‐fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA‐binding modules such as IF2‐C2, IF1 and subdomains II of elongation factors EF‐Tu and EF‐G. Structural comparisons have resulted in a model that describes the interaction between IF2‐G3 and the 30S ribosomal subunit.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.