Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy

The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site. High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence. Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base. These antibodies were reacted with 30 S ribosomal subunits from E. coli and from the chloroplasts of Alaskan peas. These two types of ribosome have been shown to be topographically similar (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers. In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform. The analogous localization in both E. coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin.     

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).     

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.     

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit

The large RNA molecule within each ribosomal subunit is folded in a specific and compact form. The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides. The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay. The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state [as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message]. Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state. Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation. Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency. Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state. With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography). We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.     

Antagonistic action between antibodies directed against 7-methylguanosine and polyamines on translation in vitro of RNA induced by measles virus

Antibodies directed against N7-methylguanosine (m7Guo) were prepared and added to a wheat germ cell-free protein-synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus. A dose-dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti-m7Guo immunoglobulins. Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG-RNA complex, no inhibition was observed.     

N4-Acetylcytidine. A previously unidentified labile component of the small subunit of eukaryotic ribosomes

The nucleoside content of 18 S rRNA from rat liver is determined under conditions known to prevent the destruction of chemically labile modified nucleosides. Two base-modified nucleosides, not completely identified before, are shown to be N6-methyladenosine and 7-methylguanosine. The results further demonstrate the presence of a hitherto unidentified component of 18 S rRNA whose spectra and chromatographic properties are identical with that of N4-acetylcytidine. In addition, this compound is not detectable in 28 S rRNA nor in 16 S rRNA derived from the small ribosomal subunit of Escherichia coli. However, it appears to be conserved in the small ribosomal subunit of eukaryotes, since it is also present in yeast 17 S rRNA and chicken liver 18 S rRNA.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

Localization of proteins S1, S2, S16 and S23 on the surface of small subunits of rat liver ribosomes by immune electron microscopy

Summary Ribosomal proteins S1, S2, S16 and S23 were localized on the surface of the small subunit (40S) of rat liver ribosomes by immune electron microscopy. Antibodies against the single proteins were raised in rabbits and chicken and purified by affinity chromatography. 40S-IgG-40S complexes were obtained by incubation of 40S subunits with non-crossreacting antibodies specific for each of the four proteins and subsequent sucrose density gradient centrifugation. The location of the proteins was determined by means of antibody binding sites visualized in negative contrast in the electron microscope. The four investigated proteins are mainly located in the head region of the small subunit. Exposed antigenic determinants of proteins S1 and S2 were found to be located at different sites of the small subunit whereas proteins S16 and S23 were mapped in a limited region only.S2,S3,S17,S21 according to the new nomenclature (McConkey et al., 1979)     

Localization of two epitopes of protein L7/L12 to both the body and stalk of the large ribosomal subunit. Immune electron microscopy using monoclonal antibodies

Four molecules of ribosomal protein L7/L12 are found as two dimers on the Escherichia coli 50 S ribosomal subunit. Immune electron microscopy using monoclonal antibodies directed against two epitopes of protein L7/L12 has allowed placement of elements of each dimer. One monoclonal antibody, directed against a determinant in the COOH-terminal domain, allows localization of two identical determinants at or near the end of the subunit stalk. The same antibody was used to place two additional determinants at the periphery of stalkless subunits, in an area from which a stalk might be expected to project. A second antibody, directed against an epitope in the amino-terminal portion of L7/L12, caused loss of stalks from the 50 S subunits. The micrographs showed symmetrical oligometric complexes of the dissociated dimeric protein with bivalent antibody. Antibodies were also seen to bind to the body of stalkless subunits, in a region near the COOH-terminal sites. The results are explained by a model in which one dimer of protein L7/L12 exists in a folded conformation on the subunit body and the second dimer occurs in an extended conformation in the subunit stalk.     

Localization of the site of cleavage of ribosomal RNA by colicin E3. Placement on the small ribosomal subunit by electron microscopy of antibody--complementary oligodeoxynucleotide complexes

Colicin E3 is a ribonuclease that inactivates Escherichia coli ribosomes by cleaving the RNA of the small ribosomal subunit after nucleotide 1493. A series of oligodeoxynucleotides that complement 16 S RNA in the region of the colicin cleavage site has been synthesized, and their ability to form complexes with 30 S ribosomal subunits has been measured using a nitrocellulose filter-binding assay. The most efficiently bound probe, complementary to residues 1485-1496, was modified with antibody-recognizable derivatives at the 5'-end, the 3'-end, or both. Antibody-oligonucleotide-subunit complexes were then generated and examined by electron microscopy. Antibody binding was seen at the tip of the platform of the 30 S subunit. The complementary oligonucleotide and thus the site at which colcin E3 cleavage occurs is therefore in the same physical region as the 3'-end of the 16 S ribosomal RNA and its message-positioning "Shine-Dal-garno" sequence.     

Influenza viral mRNA contains internal N6-methyladenosine and 5''-terminal 7-methylguanosine in cap structures.

Influenza viral complementary RNA (cRNA), i.e., viral mRNA was radioactive when purified from the cytoplasmic fraction of cordycepin-treated canine kidney cells that were incubated with [methyl-3H]methionine during infection. Approximately 55 to 60% of the methyl-3H radioactivity was in internal N6-methyladenosine, a feature distinguishing this mRNA from those viral mRNA's that are known to be synthesized in the cytoplasm. The remaining methyl-3H radioactivity was in 5'-terminal cap structures that consisted of 7-methylguanosine in pyrophosphate linkage to 2'-o-methyladenosine, N6, 2'-O-dimethyladenosine, or 2'-O-methylguanosine. Methylated adenosine was the predominant penultimate nucleoside in caps, suggesting that cRNA synthesis in infected cells initiates preferentially with adenosine at the 5' end. In contrast to cRNA, influenza virion RNA segments extracted from purified virus contained mainly 5'-terminal ppA and no detectable cap structures.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

5''-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.     

Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex

Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome. In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex). These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes. 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11. In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes. In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14. Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA.     

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.