The Escherichia coliribosomal 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 Å 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 NH4Cl 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 Å 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.

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 (F0.1% eosinophil contamination) neutrophils contained EDN, 112 6 4 ng/10 6 cells, and ECP, 163 6 2 ng/10 6 cells, whereas eosinophil major basic pro- tein (MBP) was not detectable. Electron micro- scopic 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 contami- nation) 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 neutro- phils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exer- cised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflam- mation in human disease. J. Leukoc. Biol. 63: 715-722; 1998.

ABSTRACT: 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 reagentp-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identity of the erythrocyte and placental proteins. We calculate a level of 1.5 - 3.5 x 104molecules 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.

Escherichia coli ribosomal protein L7/L12 occurs on the large subunit as two dimers: one dimer is extended and comprises the stalk, while the second dimer is folded and occupies a site on the subunit body. A variant protein, in which all 18 amino acids of the flexible hinge region that links separate N-terminal and C-terminal domains of L7/L12 has been deleted, binds the subunit as a single dimer and does not generate stalks that are visible in electron micrographs. Monoclonal antibodies directed against each domain of the protein have been used to localize the variant in electron micrographs of 50S subunits. Both C-terminal domains are seen at a shoulder of the subunit, near its edge as viewed in the most common quasisymmetric projection. N-terminal domains are placed on the subunit body, about 50 A from the C-terminal domains. The antibody to the N-terminal domain also causes dissociation of the variant dimer from the particle and the formation of oligomeric antibody-protein dimer complexes. Similar complexes were seen previously (Olson HM et al (1986) J Biol Chem 261, 6924-6936) when this antibody induced dissociation of one dimer of the native protein. We conclude that the shortened variant most probably occupies the lower-affinity site on the subunit that is normally filled by the stalk dimer.

Escherichia coli ribosomal protein L23 was derivatized with [3H]2,4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.

Pre-mRNA splicing occurs on splceosomes, 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 UI 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-Angstrom 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.

Oligodeoxynucleotide models of mRNA were used to determine the ribosomal site of specific nucleotides 3' to the initiation codon. Each mRNA analog had a 5'-terminal 9-base Shine-Dalgarno sequence, a 7-nucleotide spacer, and an ATG initiation signal, followed by up to 31 nucleotides, one of which carried an antibody-recognizable marker. All probes bound efficiently to activated Escherichia coli 30 S ribosomal subunits. Complexes were formed using ribosomal subunits, initiator tRNA, an oligodeoxynucleotide probe, and antibodies. Electron microscopy was then used to place specific positions in the mRNA analog on the subunit and thus to trace the pathway of the messenger. As viewed from the cytoplasmic side of the subunit, the 5' segment of the mRNA lies on the left, along the inner surface of the platform. The initiation codon and the next 9 nucleotides are located in the cleft between the subunit platform and body; within this segment the mRNA makes a U turn and emerges from the cleft at the left of the neck that separates the subunit head and body. The mRNA then loops around the neck to the right, along the cytoplasmic surface of the subunit and toward the site of the 7-methylguanosine residue of the 16 S ribosomal RNA.

This is the third paper in a series (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800; Olson, H. M., Olah, T., Cooperman, B. S., and Glitz, D. G. (1988) J. Biol. Chem. 263, 4801-4806) describing the use of 2,4-dinitrophenyl (DNP) derivatives of Escherichia coli 30 S ribosomal proteins to locate the positions of these proteins within the 30 S subunit by immune electron microscopy. In it we describe the derivatization of proteins S6, S13, S16, and S18 with [3H]2,4-dinitrofluorobenzene, identify the nature of the derivatized amino acids within each protein, and demonstrate that each DNP protein, denoted DNP-Sx, can be taken up into a reconstituted 30 S subunit when added to a reconstitution mixture containing 16 S rRNA and total 30 S protein depleted in Sx. We further demonstrate that each DNP-Sx binds within the 30 S subunit in a position identical or similar to that of the unmodified Sx protein, as judged by its meeting one or more of the following three criteria: (i) unmodified Sx competes with the uptake of DNP-Sx into 30 S subunits; (ii) DNP-Sx restores functional activity to those single protein omission reconstitution particles lacking full activity; (iii) DNP-Sx induces the uptake of proteins into 30 S subunits that depend on the presence of Sx. The fourth paper in this series (Montesano-Roditis, L., McWilliams, R., Glitz, D. G., Olah, T. V., Perrault, A. R., and Cooperman, B. S. (1993) J. Biol. Chem. 268, 18701-18709), which follows this one, describes the localization of the DNP-Sx proteins within the 30 S subunit by immune electron microscopy.

Purified Escherichia coli ribosomal proteins S6, S13, S16, and S18 were dinitrophenylated at their amino termini and/or at one or more internal lysine residues. Each dinitrophenyl protein was then separately incorporated into reconstituted small ribosomal subunits. Modified proteins were localized on the 30 S subunit surface by electron microscopy of reconstituted subunits complexed with antibodies to dinitrophenol (DNP). DNP protein S13 was placed on the subunit head above the platform and on the surface that faces the large subunit. DNP-S18 was localized to the subunit platform below the tip and in a region associated with binding to 50 S subunits. DNP proteins S6 and S16 were both localized near the junction of the subunit body and platform; DNP-S6 was available to antibody in 70 S ribosomes and was placed on the cytoplasm-facing side of the subunit in an area that overlaps the platform and body of the particle. DNP-S16 in 70 S ribosomes was not bound by antibody. It was localized to the 30 S body near its junction with the platform and on the surface facing the 50 S particle. The results complement and clarify data obtained using other approaches.

Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2', 3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non-secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases.

Oligonucleotides that complement Escherichia coli 16S ribosomal RNA residues 685-696 and 694-705 have been synthesized so as to incorporate antibody-recognizable markers: a 3'-terminal residue of N6-DELTA-2-isopentenyladenosine, a 5'-dinitrophenyl group, or both. Each oligonucleotide is able to bind RNA within the small ribosomal subunit, whether free or in 70S ribosomes. Immune electron microscopy places probes at nucleotides 685, 694 and 705 within a single area, at the tip of the subunit platform, very near the position of the 3'-end of the 16S RNA.

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.

Two monoclonal antibodies (mAb), directed toward different epitopes of Escherichia coli-ribosomal protein L2, have been used as probes in immune electron microscopy. mAb 5-186 recognizes an epitope within residues 5-186 of protein L2; it is seen to bind to 50 S subunits at or near the peptidyl transferase center, beside the subunit head on the L1 shoulder. mAb 187-272 recognizes an epitope within residues 187-272. This antibody binds to the face of the 50 S subunit, below the head and slightly toward the side with the stalk; this site is near the translocation domain. Both antibodies can bind simultaneously to single subunits. This indicates that protein L2 is elongated, reaching from the peptidyl transferase.center to below the subunit head and approaching the translocational domain. The different locations of the two epitopes are consistent with previous biochemical results with the two antibodies.

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.

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.