Topography of RNA in the ribosome: Localization of the 3′‐end of the 23 S RNA on the surface of the 50 S ribosomal subunit by immune electron microscopy

Shatsky, Ivan N.; Evstafieva, Alexandra G.; Bystrova, T.F.; Bøgdanov, A.; Vasiliev, V.D.

doi:10.1016/0014-5793(80)80450-9

Cited by 23 publications

(7 citation statements)

References 19 publications

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“…(6,7). The two-dimensional locations of the 3' ends of 5S and 23S RNA on 50S subunits observed by these workers were similar to those reported here [compare Fig.…”

Section: Resultssupporting

confidence: 79%

“…On the basis of these electron micrographs the 3' end of the 16S RNA has been located at the inner side of the large lobe, below the convex edge of this projection (4,5). Recently Shatsky et aL (6, 7) reported localization of the 3' ends of 50S subunit ribosomal RNAs by immunoelectron microscopy. Large ribosomal subunits were reconstituted with 5S and 23S RNA modified by covalent attachment of phenyl /3D-lactoside.…”

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confidence: 99%

See 1 more Smart Citation

Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.

Stöffler‐Meilicke¹,

Stöffler²,

Odom³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Periodate-oxidized 3' ends of 5S, 23S, and 16S rRNAs from Escherichia coli were allowed to react with fluorescein thiosemicarbazide, then labeled rRNAs were reconstituted into active ribosomal subunits. The fluorescein moiety on each of the rRNAs when reconstituted into ribosomal subunits was accessible to anti-fluorescein IgG as determined by fluorescence quenching and by sucrose gradient centrifugation. The region at which an antibody molecule bound to the labeled ribosomal subunits was determined by immunoelectron microscopy. The 3' end of the 5S RNA was localized on the central protuberance of the 50S subunit. The corresponding region for the 3' end of the 23S RNA was below the stalk on the noninterfacing surface. The 3' end of the 16S RNA was localized to the upper edge of the large lobe of 30S subunits, as reported previously.Immunoelectron microscopy has been used by three laboratories to localize the position of the 3' end of the 16S RNA in ribosomal subunits from Escherichia coli (1-5). Electron micrographs of these antibody-subunit complexes are in good agreement as to the location ofthe hapten conjugated to the 3'-terminal adenosine, although different haptens, divergent modification procedures, and even different three-dimensional models of the 30S subunits have been used by each of these groups. On the basis of these electron micrographs the 3' end of the 16S RNA has been located at the inner side of the large lobe, below the convex edge of this projection (4, 5). Recently Shatsky et aL (6, 7) reported localization of the 3' ends of 50S subunit ribosomal RNAs by immunoelectron microscopy. Large ribosomal subunits were reconstituted with 5S and 23S RNA modified by covalent attachment of phenyl /3D-lactoside.Here we present electron microscopic data localizing the 3' ends of 5S RNA and 23S RNA on reconstituted 50S ribosomal subunits containing fluorescein-derivatized RNA species that had reacted with anti-fluorescein antibody. Electron micrographs with IgG on fluorescein-labeled 16S RNA in 30S ribosomal subunits are shown for comparison with previous results (1-5).MATERIALS AND METHODS Preparation of Anti-Fluorescein Antibodies. Fluorescein isothiocyanate was conjugated to bovine serum albumin at fluorophore-to-protein ratios of 10. Three rabbits were immunized, following the immunization schedule essentially as described (8). Preimmune serum was collected prior to the first injection. IgG antibody was purified from the immune and preimmune sera by precipitation with ammonium sulfate between 0% and 50% saturation. IgG was further purified by chromatography on DEAE-cellulose columns, using conditions described by Sela and Mozes (9). The IgG fractions from rabbits 1 and 3 were further purified by affinity chromatography on staphylococcal protein A-Sepharose. IgG from any ofthe three antisera reacted with free fluorescein as well as with fluorescein-labeled ribosomal subunits. When tested by double immunodiffusion, the antisera precipitated a rabbit IgG-fluorescein conjugate, which had not been ...

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“…(6,7). The two-dimensional locations of the 3' ends of 5S and 23S RNA on 50S subunits observed by these workers were similar to those reported here [compare Fig.…”

Section: Resultssupporting

confidence: 79%

mentioning

confidence: 99%

Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.

Stöffler‐Meilicke¹,

Stöffler²,

Odom³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…8A) is in precise agreement with the position found by neutron-scattering using E. coli proteins (May et al, 1992). Furthermore, this location is close to that determined for the 3'-terminus of 23s RNA (Shatsky et al, 1980;Stoffler-Meilicke et al, 1981), and RNA-protein cross-linking and footprinting studies have both identified sites for L3 in the vicinity of the extreme 3'-terminus of the 23s molecule (see Brimacombe, 1991, for review). In the model for the arrangement of the 50s proteins proposed by Walleczek et al (1988), L3 was placed on the basis of crosslinks to proteins L13 and L19, of which only L19 had been located by IEM; this placement of L3 was rather more towards the centre of the subunit than the position shown in Fig.…”

Section: Discussionsupporting

confidence: 82%

Immunoelectron microscopic localization of ribosomal proteins BS8, BS9, BS20, BL3 and BL21 on the surface of 30S and 50S subunits from Bacillus stearothermophilus

Schwedler

Albrecht-Ehrlich

Rak

1993

European Journal of Biochemistry

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The locations of ribosomal proteins BS8, BS9 and BS20 on the 30s subunit of Bacillus stearothermophilus ribosomes, and of BL3 and BL21 on the 50s subunit, were determined by immunoelectron microscopy. BL3 was found to lie half-way down the body of the 50s subunit on the interface side, below the L7L12 stalk, in agreement with the placement of the corresponding protein in Escherichia coli by neutron-scattering; BL21 was located at a similar position on the solvent side of the subunit, as predicted by cross-linking experiments with E. coli ribosomes. Similarly, BS8 was found in the upper region of the body of the 30s subunit on the solvent side, and BS9 on the top of the head of the subunit, also on the solvent side, both positions being in good agreement with neutron-scattering data and other immunoelcctron microscopy results. In contrast, BS20 was found to lie at the extreme base of the body of the 30s subunit; this placement is not compatible with the location of E. coli S20 by neutron-scattering but fits very plausibly with other biochemical data, such as sites of RNA-protein footprinting on 16s RNA, relating to the location of S20 inImmunoelectron microscopy (IEM) is a long-established method for investigating the distribution of individual ribosomal proteins on the surface of the ribosomal subunits. In the case of the 30s subunit from Eschericlzia coli, our laboratory (Stoffler-Meilicke and Stoffler, 1990) and others (Oakes et al., 1990) have been able to locate epitopes for the majority of the small subunit ribosomal proteins with the help of this technique; the results so far have shown a high level of agreement with the neutron-scattering results of Capel et al. (1988), who have mapped the positions of the mass centers of all 21 of the 30s subunit proteins. In the case of the 50s subunit, the data sets are far less complete, although epitopes for more than half of the proteins have been located by IEM (Stoffler-Meilicke and Stoffler, 1990) and the mass centers of seven proteins have been mapped by neutron-scattering (May et al., 1992). A preliminary model for the positions of 29 of the 50s subunit proteins has been proposed (Walleczek et al., 1988), by combining the IEM data with the results of in siru protcin-protein cross-linking experiments.The more recent IEM studies have turned to the investigation of ribosomal proteins from Bacillus srearothermophi-/us. The overall morphology of ribosomal subunits from this organism is very similar to that observed in E. coli (e.g. Van Heel and Stoffler-Meilicke, 1985). Furthermore, the ribosomal proteins show a high degree of sequence similarity to their counterparts in E. coli (Wittmann-Liebold, 1988) cases so far examined it has been demonstrated that the IEM locations of similar proteins on the ribosomal subunit surfaces are identical in the two organisms (Stoffler-Meilicke et al., 1984;Stoffler and Stoffler-Meilicke, 1986; Hack1 and Stoffler-Meilicke, 1988). This finding has two important consequences. First, it shows that the sequence similarity is indeed r...

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“…2 exists in vivo [as suggested in several models for the secondary structure of 23S rRNA (9, 16)], the 5' and 3' termini of 23S rRNA can be hydrogen-bonded even in functional 50S ribosomes. Very likely, such a structure would extend out from the ribosome at the point at which the 3' terminus of 23S rRNA has been localized (19,20). Second, 50S ribosomes from strain ABL1 provide a substrate for the purification of the processing activity or activities that generate the mature termini of 23S rRNA.…”

Section: Methodsmentioning

confidence: 99%

RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA.

King¹,

Sirdeshmukh²,

Schlessinger³

1984

Proc. Natl. Acad. Sci. U.S.A.

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RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase Ill, no species cleaved by RNase Ill are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase HI were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced. rRNA in Escherichia coli is synthesized as one transcript containing 16S, 23S, and 5S rRNA as well as precursor sequences. This transcript is thought to be processed first by RNase III, which cleaves the precursor at double-stranded stems and separates the three rRNA species from one another before transcription is complete; other processing activities then remove the remaining precursor sequences during ribosome formation, generating the mature termini (1, 2). Because RNase III initiates processing, and processing has been generally considered indispensable to produce functional RNA molecules, it was unexpected that mutants deficient in the enzyme could survive. It was originally suggested that a low level of RNase III might remain in such mutants or that other RNases might form an alternative pathway to yield mature rRNA (3-5). These ideas have persisted in recent discussions, but both are disproven here in the case of 23S rRNA formation. Nuclease S1 mapping is used to show that the lesion in RNase III is complete in these mutants, but no backup pathway replaces the function of RNase III. The 16S rRNA is matured normally without any preliminary RNase III cleavage, but 23S rRNA exists only as unmatured species. Maturation of 23S rRNA is therefore absolutely dependent on RNase III action, but at least some of the unmatured 23S rRNA species detected must function in protein synthesis because these mutant strains are viable. The maturation of the unprocessed 23S rRNA in 50S ribosomes is shown to occur in subcellular extracts of a RNase III+ strain. MATERIALS AND METHODSThe termini of rRNA species in two RNase III-deficient strains, ABL1 and AB301-105 (6, 7), were assessed by their capacity to hybridize to DNA probes composed of complementary sequences of rDNA. ABL1 and AB301-105 cells were grown in Luria broth at 30°C to an optical density of 0.25 at 550 nm and then harvested over ice. Total RNA was prepared by extracting cells directly at 65TC with phenol in the presence of 1% sodium dodecyl sulfate. The RNA was precipitated once with LiCI (8) and twice with ethanol and was then suspended at a standard concentration. Singlestranded DNA hybridization probes for each termi...

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Topography of RNA in the ribosome: Localization of the 3′‐end of the 23 S RNA on the surface of the 50 S ribosomal subunit by immune electron microscopy

Cited by 23 publications

References 19 publications

Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.

Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.

Immunoelectron microscopic localization of ribosomal proteins BS8, BS9, BS20, BL3 and BL21 on the surface of 30S and 50S subunits from Bacillus stearothermophilus

RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA.

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