Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling.

The Escherichia coli ribosomal protein L7/L12 is central to the translocation step of translation, and it is known to be flexible under some conditions. The assignment of electron density to L7/L12 was not possible in the recent 2.4 A resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920). We have localized the two dimers of L7/L12 within the structure of the 70 S ribosome using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction. First, the structures were determined for ribosomal cores from which protein L7/L12 had been removed by treatment with NH(4)Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11. Second, ribosomes were reconstituted using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 A gold derivative. The reconstruction from cryo-electron microscopy images and difference mapping placed the gold at four interfacial positions. The finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of elongation and translocation.

Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.

Ribonuclease inhibitor protein of human erythrocytes: characterization, loss of activity in response to oxidative stress, and association with Heinz bodies.

Significant amounts of ribonuclease inhibitor protein are present in human and rat erythrocytes, cells that are essentially devoid of ribonuclease or functional RNA. The protein from human erythrocytes is indistinguishable from human placental ribonuclease inhibitor protein by immunological and biochemical criteria. Each inhibitor forms an equimolar complex with bovine pancreatic ribonuclease A and is inactivated by treatment with the sulfhydryl reagent p-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identify of the erythrocyte and placental proteins. We calculate a level of 1.5-3.5 x 10(4) molecules of active inhibitor per erythrocyte, most or all of which occurs in an uncomplexed form since inactivation of the inhibitor revealed barely detectable levels of RNase activity. Immunogold localization showed a high level of labeling and a uniform distribution of gold particles in the cytoplasm of erythrocytes, while little inhibitor activity was found in association with isolated red blood cell membranes. Oxidative stress on isolated red cells resulted in a decrease in the level of reduced glutathione and a gradual and irreversible loss of inhibitor activity; inhibitor disappeared from the cytosol and became associated with nascent Heinz bodies. We suggest a role for this protein in the metabolism and aging process of the erythrocyte.

Purification of the spliceosome A-complex and its visualization by electron microscopy.

Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit.

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.

Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody.

A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy. The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes. It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+. Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1. There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities. Fab fragments of the antibody gave the same result as the intact antibody. Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding. Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits. This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface.

Localization of an oligodeoxynucleotide complementing 16S ribosomal RNA residues 520-531 on the small subunit of Escherichia coli ribosomes: electron microscopy of ribosome-cDNA-antibody complexes.

The oligodeoxynucleotide dACCGCGGCTGCT, complementary to Escherichia coli small ribosomal subunit RNA residues 520-531, has been used to probe subunit conformation and to localize the sequence in the subunit. Conditions for binding of the cDNA to 30S subunits were optimized and specificity of the interaction was demonstrated by RNase H cleavage. Three kinds of terminal modification of this cDNA were used to allow its localization by immune electron microscopy. A solid phase support with 5'-dimethoxytrity-N6-delta 2-isopentenyl-adenosine linked to controlled pore glass was synthesized, and used to prepare oligomer with an added 3'-terminal residue of isopentenyl adenosine. cDNA with a 5' primary amine substituent was modified with 1-fluoro-2,4-dinitrobenzene to prepare 5'-dinitrophenyl oligonucleotide, and both modifications together gave doubly-derivatized probes. Immune electron microscopy with antibodies to dinitrophenol, isopentenyl adenosine, or both, was used to place the cDNA on 30S subunits. In each case the probe was placed at a single site at the junction of the head and body of the subunit, near the decoding site and the area in which elongation factor Tu is bound. It is proposed that this segment of ribosomal RNA functions in mRNA binding and orientation.

Purification and characterization of a ribonuclease from human liver.

The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.

Amino acid sequence of the nonsecretory ribonuclease of human urine.

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)

Wheat germ cytoplasmic ribosomes. Structure of ribosomal subunits and localization of N6,N6-dimethyladenosine by immunoelectron microscopy.

Cytoplasmic ribosomes have been isolated from wheat germ, and the structure of ribosomal subunits has been examined by electron microscopy of negatively stained preparations. Small (40 S) subunits show structural features generally regarded as characteristic of eukaryotic particles, while large (60 S) subunits show shapes that are equally well described by models of prokaryotic 50 S particles. Small subunit 18 S RNA contains 2 residues of N6,N6-dimethyladenosine 19 and 20 residues from the 3'-end (Hagenbüchle, O., Santer, M., Steitz, J. A., and Mans, R. J. (1978) Cell 13, 551-563). Nucleoside analysis by high performance liquid chromatography shows no other residues of this component in the RNA. Anti-dimethyladenosine immunoglobulins were reacted with wheat germ 40 S subunits, and the resulting complexes were studied by electron microscopy in order to localize the nucleoside. In about 90% of the complexes observed, antibody-subunit contact was consistent with a single binding site. We place the dimethyladenosine residues at or near the end of the platform of the 40 S particle in a position nearly equivalent to that previously identified in prokaryotic and chloroplast subunits (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879).

Wheat germ cytoplasmic ribosomes. Localization of 7-methylguanosine and 6-methyladenosine by electron microscopy of immune complexes.

Nucleoside analysis of the RNA from the small subunit of wheat germ cytoplasmic ribosomes shows 1 mol each of N7-methylguanosine and N6-methyladenosine/mol of RNA. Antibodies directed against each methylated nucleoside were used to localize these residues within the subunit by electron microscopy of immune complexes. Antibodies to 7-methylguanosine bound 40 S subunits at a single site, at or slightly above the division between the upper and lower segments of the particle and on the surface furthest from the platform (or large lobe) of the subunit. This site is essentially equivalent to that previously seen with Escherichia coli and chloroplast 30 S subunits (Trempe, M. R., Ohgi, K., and Glitz, D. G. (1982) J. Biol. Chem. 257, 9822-9829). Antibodies to N6-monomethyladenosine were induced in rabbits with a nucleoside-albumin conjugate and shown to be specific for the modified nucleoside. Electron microscopy of antibody-subunit complexes placed the methyladenosine residue in a position that is essentially indistinguishable from that of 7-methylguanosine.

Probing ribosome function and the location of Escherichia coli ribosomal protein L5 with a monoclonal antibody.

A monoclonal antibody specific for Escherichia coli ribosomal protein L5 was isolated from a cell line obtained from Dr. David Schlessinger. Its unique specificity for L5 was confirmed by one- and two-dimensional electrophoresis and immunoblotting. The antibody recognized L5 both in 50 S subunits and 70 S ribosomes. Both antibody and Fab fragments had similar effects on the ribosome functions tested. Antibody bound to 50 S subunits inhibited their reassociation with 30 S subunits at 10 mM Mg2+ but not 15 mM, the concentration present for in vitro protein synthesis. The 70 S couples were not dissociated by the antibody. The antibody caused inhibition of polyphenylalanine synthesis at molar ratios to 50 S or 70 S particles of 4:1. The major inhibitory effect was on the peptidyltransferase reaction. There was no effect on either elongation factor binding or the associated GTPase activities. The site of antibody binding to 50 S was determined by electron microscopy. Antibody was seen to bind beside the central protuberance or head of the particle, on the side away from the L7/L12 stalk, and on or near the region at which the 50 S subunit interacts with the 30 S subunit. This site of antibody binding is fully consistent with its biochemical effects.

Immunological assay of pancreatic ribonuclease in serum as an indicator of pancreatic cancer.

Serum levels of RNase activity, presumed to originate in the pancreas, have been suggested to be of use in the diagnosis of pancreatic cancer. We have used a radioimmunological assay of human pancreatic-like RNase to quantitate this protein in serum from normal blood donors and patients with a variety of diseases. Serum pancreatic-like RNase rises gradually with age, and its level is usually higher in males than females. Although many patients with pancreatic cancer show elevated serum levels of immunologically cross-reactive enzyme, others are apparently normal. In several other types of cancer, a similar pattern of elevated RNase is apparent. However, in kidney or bladder carcinoma and in patients with severe kidney disease, RNase levels are almost always greater than normal. Regardless of the nature of the disease, an elevated level of pancreatic-like enzyme is usually accompanied by above-normal levels of serum urea nitrogen. Hence, elevated circulating levels of pancreatic-like RNase are best related to kidney function and do not serve as a specific marker for cancers of the pancreas or other organs.

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations.

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.

Chloroplast ribosome structure. Electron microscopy of ribosomal subunits and localization of N6,N6-dimethyladenosine by immunoelectronmicroscopy.

Ribosomal subunits from the chloroplasts of Alaskan peas have been studied by immunoelectronmicroscopy. Electron micrographs of negatively stained small and large ribosomal subunits show particles of similar size and in the same characteristic projections described for the ribosomal subunits of Escherichia coli (Lake, J. A. (1976) J. Mol. Biol. 105, 131-159), although minor structural differences are apparent. High pressure liquid chromatographic analysis shows the modified nucleoside N6,N6-dimethyladenosine is conserved in chloroplast 16 S ribosomal RNA, presumably as two successive residues near the 3' end. Antibodies directed against N6,N6-dimethyladenosine were allowed to react with chloroplast 30S ribosomal subunits. Electron microscopy showed individual subunit-antibody complexes and pairs of ribosomal subunits cross-linked by a single antibody. In 94% of the complexes observed, antibody contact was consistent with a dimethyladenosine localization near the end of the small subunit platform, in an area of subunit contact in the 70 S ribosome. This localization is analogous to the placement of N6,N6-dimethyladenosine in the E. coli ribosome (Politz, S. M., and Glitz, D. G. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1468-1472).

Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6, N6-dimethyladenosine.

The modified nucleoside N6, N6-dimethyladenosine occurs in Escherichia coli 16S ribosomal RNA only in two successive positions near its 3' end. Antibodies directed against dimethyladenosine were induced with a nucleoside-albumin conjugate. As measured by second antibody precipitation of immune complexes, antidimethyladenosine antibodies bound 30S ribosomal subunits, ribosomal core particles, and ribosomal RNA which contain dimethyladenosine but showed little cross-reactivity with RNA or ribosomal subunits from a kasugamycin-resistant mutant which lacks dimethyladenosine. Antibody binding to ribosomal subunits was strongly influenced by the concentration of magnesium ion in the reaction medium and by the prior treatment of the subunits. Functionally active 30S subunits showed a striking binding optimum at 2-4 mM Mg2+; this optimum disappeared if the subunits were inactivated by dialysis against low concentrations of magnesium ion. Instead, the inactivated subunits showed a gradual increase in antibody binding as the magnesium ion concentration was raised to 20 mM; binding of 16S ribosomal RNA or subribosomal core particles from 30S subunits gave qualitatively similar curves, with no evidence of a low [Mg2+] optimum. The stability of antibody-subunit complexes was also found to depend upon subunit conformation and magnesium ion concentration; the half-life of an inactivated subunit-antibody complex (15 mM Mg2+) averaged 130 min, while active subunit-antibody complexes (3 mM Mg2+) had an average half-life of 70 min. More of the immune complexes with inactivated subunits were found to survive sucrose gradient sedimentation (relative to active subunits), and the concentration of subunits needed to halve antibody binding of [3H]-N6, N6-dimethyladenosine was lower with inactivated subunits. The results suggest that the antibody binding optimum seen with active subunits at 2-4 mM Mg2+ represents a dynamic aspect of the three-dimensional ribosomal subunit structure; a site near the 3' end of the RNA is involved, and both the availability of the modified nucleoside to an antibody probe and the stability of the resulting complexes are involved.

Localization of the site of adenylylation of glutamine synthetase by electron microscopy of an enzyme-antibody complex.

Antibodies to the nucleosidel,N(6)-ethenoadenosine have been used to localize the site of adenylylation of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Escherichia coli. Antibodies were induced in rabbits by injection of a bovine albumin-ethenoadenosine conjugate. The resulting antisera strongly bound ethenoadenosine, its 5'-nucleotide, or protein conjugates of the nucleoside; little or no crossreaction was seen to adenosine, AMP, or the protein carrier. Ethenoadenylylated glutamine synthetase was prepared by modification of the enzyme by the E. coli adenylyltransferase, using etheno-ATP as a substrate. The ethenoadenylylated glutamine synthetase was precipitated by antibodies to ethenoadenosine in conjunction with goat anti-rabbit gamma globulin. Electron micrographs of reaction mixtures of ethenoadenylylated glutamine synthetase and anti-ethenoadenosine showed individual enzyme molecules complexed with one or more antibodies and pairs of enzyme molecules crosslinked by a single antibody. The approximate site of adenylylation was located from the apparent area of contact between enzyme and antibody. We conclude that the adenylylation sites are on the periphery of the bilayered hexagonal disc, offset by 15 +/- 10 degrees from the 2-fold axis of symmetry through a vertex of the hexagon and 20 +/- 10 A from the plane between the layers of the disc.

Antibodies to N6-(delta2-isopentenyl) adenosine and its nucleotide: interaction with purified tRNAs and with bases, nucleosides and nucleotides of the isopentenyladenosine family.

The interaction of antibodies directed toward N6-(delta2-isopentenyl)adenosine, i6Ado, or its nucleotide with related bases, nucleosides, nucleotides and purified tRNAs is described. The selectivity of the antibody preparation was tested in inhibition experiments utilizing a sensitive radioimmunoassay to quantitate the binding of [3H]i6Ado to the antibody. Purified tRNAs containing various modified nucleosides adjacent to the 3'-end of the anticodon were tested to provide information about the selectivity of the antibody preparation toward nucleotides in this position of the tRNA chain. Antibodies directed against the nucleotide hapten were used to purify tRNAs which contain i6Ado and to quantitate the amount of that nucleotide. The same order of selectivity was expressed whether the nucleotides were free or in a tRNA molecule. Interaction of the antibody with compounds from the i6Ado family demonstrated dominance of the hydrophobic isopentenyl group and the importance of positional differences of modifications.

Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex.

Antibodies to the minor nucleoside N6,N6-dimethyladenosine have been used to map a unique location of the nucleoside in the small subunit of the Escherichia coli ribosome. Antibodies were induced in rabbits by a nucleoside-bovine albumin conjugate and shown to be highly specific for the dimethyladenosine hapten. The antibodies were shown to interact with 30S ribosomal subunits from strain PR7, but not with subunits from its mutant strain TPR201, which is resistant to kasugamycin and lacks the two successive residues of dimethyladenosine normally found near the 3'-end of E. coli 16S ribosomal RNA. Electron micrographs of strain PR7 subunits, crosslinked by single IgG molecules, show a single binding site on the surface of the ribosome. This binding site is consistent with observations relating the 3'-end of the ribosomal RNA, binding of initiation factor IF-3 and messenger RNA, and mapping of specific ribosomal proteins.