Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

Small-angle X-ray scattering study of ribosomal proteins S3, S4 S7 and s20.

Ribosomal proteins S3, S4, S7, S20 from Escherichia coli have been studied by small-angle X-ray scattering techniques. The molecular weights found by X-ray scattering agree with other determinations. The large values of the radii of gyration indicate anisometric particles. A more detailed morphological analysis is hindered by low solubility. An interpretation of the experimental results is given in terms of compact objects of simple shape. Yet the possibility is envisaged that these proteins may be less rigid and compact than other proteins.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.     

Analysis of models of irregular shape by solution X-ray scattering: the case of the 50S ribosomal subunit from E. coli

Structural models of biological macromolecules can be tested by comparing calculated and experimental solution scattering curves. We have developed an approach for computing scattering shape functions at medium resolution from models proposed on the basis of other techniques such as electron microscopy. We present the results obtained with the 50S ribosomal subunit from Escherichia coli; two models are considered, one proposed by Lake (1976), the other one by Tischendorf et al. (1975). Although the two models are similar in many respects, their scattering shape functions are significantly different. The comparison with the experimental scattering curve allows us to check the scale of the models and, after scaling, to quantitate the agreement between the observed and the calculated curves. Finally, it can provide a starting point for the structural interpretation of the X-ray data.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

Small-angle X-ray titration of the complex formed between the ribosomal protein S4 and its 16S binding site, S4-RNA: a central core in the 30S subunit.

X-ray scattering titrations at 21 degree C and in ribosomal reconstitution buffer indicate that the S4-RNA and the protein S4 from a 1:1 complex with a stability constant, log K approximately 6.5. When the complex forms, there is only a limited change in the scattering curve indicating that S4-RNA essentially retains its conformation during the complex formation. The increase in the gyration radius as a result of the complex formation, delta R = 4 +/- 3 A, as well as the experimental scattering curve of the complex can be explained by models where the protein S4 is supposed to interact with the periphery of the S4-RNA.     

Determination of the distribution of protein and nucleic acid in the 70 S ribosomes of Escherichia coli and their 30 S subunits by neutron scattering.

Neutron small-angle scattering of the 70 S Escherichia coli ribosomes and of its smaller 30 S subunit has been measured in $\text{H}{}_2\text{O}{}^2\text{H}{}_2\text{O}$ mixtures. A linear dependence of the square of the radius of gyration on the reciprocal of the contrast is found, which is qualitatively similar to the results from contrast variation with the larger 50 S subunit. The slope α in this plot is a measure of radial segregation of RNA and proteins. It is most pronounced with the 50 S subunit. The 30 S particle appears to be more homogeneous, whereas the 70 S ribosome assumes an intermediate value of α. Neither the 30 S and 50 S subunits nor the 70 S ribosome show a significant separation of the centres of mass of their RNA part and proteins. A quantitative comparison of the parameters obtained suggest that the interaction between the two subunits and the 70 S ribosomes does not involve any major change in the latter.     

Neutron-scattering studies of the ribosome of Escherichia coli: a provisional map of the locations of proteins S3, S4, S5, S7, S8 and S9 in the 30 S subunit.

Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed.     

The alkenic unreactivity of mono- and bi-cyclic derivatives of 3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide

The inertness of the alkenic bond towards electrophilic additions in 3-exocyano-3-(methylthio)-2-thiabicyclo[2.2.1]hept-5-ene S,S,S′,S′-tetraoxide (5), 3,6-dihydro-2-(methylthio)-2H-thiopyran-2-carbonitrile S,S,S′,S′-tetraoxide (3), and 2-(acetamidomethyl)-3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide (4) is attributed to the “supra-annular effect” and field effects. Conformational analysis of a pentadeuterated derivative of 4 (10) is reported. On the basis of the 220-MHz ¹H n.m.r.-spectral data of 10, the compound was concluded to adopt the ⁰H₂ conformation in chloroform solution.     

Methylated 23S rRNA nucleotide m2G1835 of Escherichia coli ribosome facilitates subunit association

Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m²G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal titration calorimetry and Rayleigh light scattering, we have found that methylation of m²G1835 specifically enhances association of ribosomal subunits. No defects in fidelity of translation or interaction with translation GTPases could be ascribed to the ribosomes unmethylated at G1835 of the 23S rRNA. Methylation of G1835 was found to provide a significant advantage for bacteria at osmotic and oxidative stress.     

TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis,S. Typhimurium,S. Infantis,S. Virchow,S. Hadar,S. Newport and S. Anatum

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N₆(GTG)₄, N₆(CAC)₄, N₆(CGG)₄, N₆(CCG)₄ and N₆(CTG)₄, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N₆(GTG)₄ primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.     

Structural studies on the 30 S ribosomal subunit from Escherichia coli

Small-angle X-ray scattering curves computed for various 30 S subunit structures have been compared with the experimental scattering curve. The curve from the 30 S subunit is best approximated by that calculated for a 1:3.6:3.6 ellipsoidal structure. The rather prolate ellipsoidal model suggested by recent electron microscope studies gives a scattering curve considerably different from the 30 S curve, suggesting that the electron microscope model is not that found in solution. Analysis of the more extended portions of the experimental scattering curve suggests some internal structure. A model is proposed that contains RNA and protein in positions such that the calculated scattering curve shows more extensive, yet similar internal structure. Resultant constraints on the structure of the 30 S subunit in solution are given.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

X-ray scattering of antibodies: the monomeric 8S subunit of human IgM

8S monomeric subunit of a human immunoglobulin M was investigated by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 5.9 nm, maximum length 21 nm, hydrated volume 410 nm³ and two radii of gyration of the cross-section: 2.6 and 1.8 nm. A model equivalent in scattering was found and compared with the model for a human IgG also based on small-angle X-ray scattering data. The Cμ2 domain of the IgM obviously has a very loose structure, and the Fab angle of the 8S IgM (90°) is smaller than that of the IgG (134°).     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: The N-terminal region of S2 facilitates self-association

The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo.     

Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10⁻⁷ M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.