Preparation of a ''beheaded'' derivative of the 30S ribosomal subunit

In order to test the hypothesis that an alteration in the sex hormone milieu may underlie risk factors for myocardial infarction, fasting serum sex hormones, ie, estradiol, testosterone, free testosterone, and androstenedione, were measured in 24 hypertensive and in 19 healthy postmenopausal women. The mean serum free testosterone level (P=0.01) and the free-to-total testosterone ratio (P < 0.04) were increased in the women with hypertension. In a stepwise multiple regression analysis on the hypertensive and normotensive groups combined, with systolic blood pressure (SBP) as the dependent variable and body mass index, age, free testosterone, estradiol, insulin, and cholesterol levels as the independent variables, only free testosterone showed an independent relationship to SBP (P=0.009). The finding in the present study of an independent positive relationship of free testosterone with hypertension is consistent with a similar relationship of free testosterone with other risk factors for myocardial infarction in women found in previous studies and supports the hypothesis.     

Macrolide antibiotics inhibit 50S ribosomal subunit assembly in Bacillus subtilis and Staphylococcus aureus

Macrolide antibiotics are clinically important antibiotics which are effective inhibitors of protein biosynthesis in bacterial cells. We have recently shown that some of these compounds also inhibit 50S ribosomal subunit formation in Escherichia coli. Now we show that certain macrolides have the same effect in two gram-positive organisms, Bacillus subtilis and Staphylococcus aureus. Assembly in B. subtilis was prevented by erythromycin, clarithromycin, and azithromycin but not by oleandomycin. 50S subunit formation in S. aureus was prevented by each of seven structurally related 14-membered macrolides but not by lincomycin or two streptogramin antibiotics. Erythromycin treatment did not stimulate the breakdown of performed 50S subunits in either organism. The formation of the 30S ribosomal subunit was also unaffected by these compounds. Assembly was also inhibited in a B. subtilis strain carrying a plasmid with the ermC gene that confers macrolide resistance by rRNA methylation. These results suggest that ribosomes contain an additional site for the inhibitory functions of macrolide antibiotics.     

In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement

Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in complexity, and many of their subunits display a high degree of sequence similarity to their eukaryotic counterparts. Here we describe specific protein-protein contacts present between individual recombinant RNAP subunits from the archaeon Methanococcus jannaschii. Subunits D and L interact specifically with each other in two-hybrid assays. D also interacts under the same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces cerevisiae, suggesting that essential elements of the binding surface between these proteins have been conserved across the archaeal/eukaryotic evolutionary domain boundary. Interactions between L and RPB3 or AC40 were, however, not detectable. Recombinant D and L subunits associate under in vitro conditions and copurify with each other during size-exclusion chromatography. Addition of an another recombinant subunit (N) to the D-L complex results in the formation of a triple complex. This D-L-N complex resembles the RPB3-RPB11-RPB10 or AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively. Our data provide evidence for a close similarity in the quaternary arrangement of a subset of archaeal and eukaryotic RNA polymerase subunits and the conservation of the protein-protein contacts formed between them.     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.     

Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure

L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.     

Translocation of 60S ribosomal subunit in spreading cardiac myocytes

Cardiac myocytes in culture undergo considerable structural reorganization. The remodeling of the myofibrils and the nonmyofibrillar cytoskeleton that occurs in the spreading cardiac myocytes resembles the cellular features observed in the hypertrophying heart. In this study we examined the distribution of the large 60S ribosomal subunit in freshly isolated cardiac myocytes and during the course of attachment and spreading in culture. Initially, anti-60S immunolabeling was scattered widely throughout the sarcoplasm of the dissociated cardiac myocytes. After attachment to the substrate, the 60S ribosomal subunit attained wide sarcoplasmic localization before a sarcomere-related staining pattern appeared in the spreading cell. Double labeling experiments with alpha-actinin confirmed co-localization of the 60S ribosomal subunit with nascent and mature myofibrils. These findings demonstrate that translocation of the 60S ribosomal subunit coincides with the cytoskeletal reorganization taking place in these cells. Moreover, the close association between the myofibrils indicates a particular role for the ribosomes in maintenance and growth of the contractile apparatus. (J Histochem Cytochem 46:963-969, 1998)     

In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum

In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.     

In vitro assembly and disassembly of coatomer

Coatomer, a complex of seven proteins, is the major component of the non-clathrin (COP I) membrane coat. We report here the first system to reversibly disassemble and reassemble this complex in vitro. Coatomer disassembles at high salt concentrations and reassembles when returned to a more physiological buffer. Using this system, we show that alpha-, beta'-, and epsilon-COP interact directly and that gamma-COP interacts with zeta-COP. A partial complex comprising alpha-, beta'-, and epsilon-COP, obtained after coatomer disassembly, can bind to membranes in vitro. This binding is, at least in part, mediated by interactions with cytoplasmic KKXX motifs of proteins normally retained in or retrieved to the endoplasmic reticulum. Using coatomer disassembly and epitope-specific antibodies, we also demonstrate that the N- and C-terminal domains of beta-COP are buried within the native coatomer complex. These results provide the first insights into how the coatomer is structured.     

Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit

It is now generally accepted that 16S and 23S ribosomal RNA play important roles in the decoding and peptidyl transferase activities of ribosomes. Despite their complex structures and numerous associated proteins it is possible that small domains of these rRNAs can fold and function autonomously, particularly those that appear devoid of protein interactions. One candidate for such a domain is the decoding region, located near the 3' end of 16S rRNA (Fig. 1a, b). Consistent with this hypothesis, aminoglycoside antibiotics that interact with the decoding region in 30S subunits interact with other RNAs in the absence of proteins. In addition, certain activities of self-splicing introns, at least superficially, resemble translational decoding. We report here that an oligoribonucleotide analogue of the decoding region interacts with both antibiotic and RNA ligands of the 30S subunit in a manner that correlates with normal subunit function. The activities of the decoding region analogue suggest that the intimidating structural complexity of the ribosome can be, to some degree, circumvented.     

Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication

The recent development and use of protease inhibitors have demonstrated the essential role that combination therapy will play in the treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1). Past clinical experience suggests that due to the appearance of resistant HIV-1 variants, additional therapeutics will be required in the future. To identify new options for combination therapy, it is of paramount importance to pursue novel targets for drug development. Ribosomal frameshifting is one potential target that has not been fully explored. Data presented here demonstrate that small molecules can stimulate frameshifting, leading to an imbalance in the ratio of Gag to Gag-Pol and inhibiting HIV-1 replication at what appears to be the point of viral particle assembly. Thus, we propose that frameshifting represents a new target for the identification of novel anti-HIV-1 therapeutics.     

Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits: a functional role for the N-terminus of the factor

Malignant histiocytic disorders, other than leukemias, are extremely rare in childhood. Despite unresolved nosologic and terminologic difficulties, they should be classified according to the lineage of the aberrant cells in a given tumor. There are no common and typical clinical presentations, nor are there established treatment modalities available. For the disseminated forms, aggressive systemic treatment modalities--similar if not identical to those used for large cell anaplastic lymphomas--appear to be the best treatment option. For the localized forms, which are primarily dendritic cell sarcomas, a more localized and individualized therapy is appropriate.     

Azithromycin and clarithromycin inhibition of 50S ribosomal subunit formation in Staphylococcus aureus cells

A mercapturic acid attached to the aromatic ring of toluene was for the first time detected in human urine as a metabolite of toluene. Since the metabolism of toluene is usually considered to take place at the side-chain, this gives, besides the biosynthesis of cresols, a further hint of a metabolic conversion of the aromatic system. We examined a group of 33 workers occupationally exposed to toluene, determining the concentrations of toluene in ambient air and in whole blood, o-cresol and hippuric acid in urine and p-toluylmercapturic acid (p-TMA) in urine. All blood and urine samples were collected post-shift. The renal excretion of S-p-toluylmercapturic acid showed highly significant correlations with established parameters of a biological monitoring of toluene. The median ambient air concentration was 63 ppm, ranging from 13 to 151 ppm, the median concentration of toluene in whole blood was 804 microg/l, corresponding to median urinary concentrations for o-cresol of 2.3 mg/l, hippuric acid of 2.3 g/l and p-TMA of 20.4 microg/l. p-TMA was not detectable in urine samples of a control group of 10 non-exposed persons. Both the German Biological Tolerance Values (BAT-values) for toluene in blood (1000 microg/l) and o-cresol in urine (3 mg/l) correspond to a mean p-TMA elimination of approximately 50 microg/l, and thus are in agreement with each other. According to our results p-TMA reflects internal toluene exposure diagnostically sensitive and specifical. With the developed analytical procedure we determined a median benzylmercapturic acid (BMA) concentration of 190 microg/l in the urine samples of the toluene exposed persons. We also determined a median BMA concentration of 30 microg/l in the control samples of non-exposed persons. However, these results are preliminary and require further confirmation as the reliability of the method was determined only for p-TMA.     

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit

Biotinylation has become a popular alternative to radioiodination for labeling cell surface proteins, whereas labeling of the total cellular protein pool is usually achieved metabolically with [35S]methionine and [35S]cysteine. In this paper we describe a new technique in which total cellular lysate proteins that have been affinity bound to a solid phase are labeled efficiently with biotin. This labeling technique is preferable to direct biotinylation of cell lysate since the unreacted biotin can be readily removed from the sample by washing. The affinity step permits preselection of the molecules to be labeled, thereby decreasing the potential for nonspecific binding during subsequent immunoprecipitation. We applied this affinity biotinylation method to a human cellular lysate in order to preselect the total glycoprotein pool for subsequent immunoprecipitation of HLA class I. Following immunoprecipitation, SDS-PAGE, and Western blot, the biotinylated protein could be readily revealed by enhanced chemiluminescence. The results were comparable to those obtained by radiometabolic labeling and Western blot using a monoclonal antibody probe. Overall, the affinity biotinylation method is faster and more practical than conventional radiolabeling, without any loss in sensitivity.     

In vitro reconstitution of an intermediate assembly stage of vaccinia virus

The effects of chronic exogenous testosterone treatment on the synthesis and/or secretion of two sturgeon gonadotropins (stGTH I and stGTH II) were assessed in 2-year-old juvenile white sturgeon (Acipenser transmontanus) surgically implanted with silastic capsules filled with 75 mg of testosterone and in previtellogenic female white sturgeon females implanted with 150 mg of testosterone. In groups of juvenile white sturgeon sacrificed 30, 60, 90, or 442 days postimplantation, pituitary concentrations of stGTH I were significantly greater in testosterone-treated fish (P < 0.01) when compared to those of controls. Pituitary concentrations of stGTH II were significantly higher (P < 0.01) in juvenile fish treated 60, 90, or 442 days with testosterone when compared to those of controls. Exogenous testosterone had no effect on plasma concentrations of either stGTH. Additional testosterone-treated juvenile sturgeon which were injected intraperitoneally 90 or 442 days postimplantation with 10 microg/kg of the gonadotropin releasing hormone analog d-Ala6-des-Gly10-GnRH ethylamide (GnRHa) also showed no change in plasma concentrations of stGTHs. Similar results were obtained for previtellogenic white sturgeon, as pituitary concentrations of stGTH I and stGTH II were significantly greater (P < 0.01) after 60 days of testosterone treatment compared to those of controls. A second group of 60-day testosterone-treated previtellogenic females also failed to exhibit increases of plasma stGTHs when administered 10 microg/kg of GnRHa. These results indicate that long-term testosterone treatment stimulates the accumulation of pituitary stGTHs in both juvenile and previtellogenic white sturgeon but does not affect basal or GnRHa-induced stGTH secretion.     

Database on the structure of large subunit ribosomal RNA

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/     

Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.