Evolving Ribosome Structure and Function: rRNA and the Translation Mechanism

Oakes, Melanie; Scheinman, A.; Rivera, Maria C.; Soufer, D.; Shankweiler, G.; Lake, James A.

doi:10.1101/sqb.1987.052.01.077

Cited by 4 publications

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“…Much of the delineation of such domains resulted from site mapping experiments, which to a large extent have not been carried out for the eukaryotic ribosome. Two notable exceptions are the mapping of the exit site for both eubacterial and eukaryotic ribosomes by Bernabeau and coworkers (1983), and the mapping of the site of the P-site anticodon-codon interaction (Oakes et al, 1987;cf. Ciesiolka et al, 1985).…”

Section: Comparison To Coo E Coli Reconstructionmentioning

confidence: 99%

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome.

Verschoor

Srivastava

Grassucci

et al. 1996

The Journal of Cell Biology

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Abstract. A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen-hydrated electron microscopic preparation reveals the native structure of this macromolecular complex. The new structure, at 38 A resolution, shows a marked resemblance to the structure determined for the E. coli 70S ribosome (Frank, J., A. Verschoor, Y. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, and S. Srivastava. 1996b. In press; Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava, A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, and R. Agrawal. 1995. Nature (Lond.). 376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed. Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small-subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet. However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure. The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea). The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit. This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface.THOUGH much less well studied than the E. coli 70S ribosome, largely because of its greater complexity and difficulty of preparation, the 80S eukaryotic ribosome shows a great deal of similarity to its eubacterial counterpart. Increases in numbers of components, both ribonucleoprotein (RNP) 1 components and nonribosomal translational factors (for review see Nygard and Nilsson, 1990), are thought to be the result of the need for greater accuracy of the translational process, and tighter regulation of the steps involved in it, but in general the eukaryotic translational process shows strong homology with the eubacterial one.The eukaryotic ribosome is a highly intricate macromolecular complex. It is significantly larger than the eubacterial ribosome, with a molecular mass of ~4 million daltons, as compared to 2.8 million daltons. Across the kingdom Eukarya, however, the large (60S) in size. In plants it is 2.45-2.5 million daltons, while in mammals it may reach 3 million daltons (Bielka, t982). Some of the size variability of the 60S subunit is due to variation in the size of the 28S rRNA, which ranges from 1.2 to 1.7 million daltons (Bielka, 1982). In contrast, the molecular mass of the small (40S) subunit is fairly constant, ~1.5 milhon daltons.In the 40S ribosomal subunit, the ...

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Section: Comparison To Coo E Coli Reconstructionmentioning

confidence: 99%

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome.

Verschoor

Srivastava

Grassucci

et al. 1996

The Journal of Cell Biology

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“…In this 9-base loop in the central domain of 16S rRNA, 6 of the bases are universally conserved and 2 additional bases are strongly but not universally conserved (6). The loop has been localized to the platform of the subunit in the area that interfaces with the 50S subunit (3,4,7). Several studies have established that many of the bases in the 790 loop are exposed in 30S subunits and inaccessible in 70S ribosomes (8)(9)(10)(11)(12)(13).…”

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confidence: 99%

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

Tapprich

Goss

Dahĺberg

1989

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

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A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia colimrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affiity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.Several regions of rRNA have been shown to be involved in the process of translation, primarily regions that are single stranded and highly conserved phylogenetically (1,2). The locations of these sequences in Escherichia coli 16S rRNA have now been placed in three-dimensional models ofthe 30S ribosomal subunit (3, 4). In general there is good agreement between structure and function since many single-stranded highly conserved rRNA regions, proposed to carry out related functions in translation, are clustered in the same area of the subunit (5).One example of a region with a structural placement consistent with its proposed function is the 790 loop of 16S rRNA. In this 9-base loop in the central domain of 16S rRNA, 6 of the bases are universally conserved and 2 additional bases are strongly but not universally conserved (6). The loop has been localized to the platform of the subunit in the area that interfaces with the 50S subunit (3, 4, 7). Several studies have established that many of the bases in the 790 loop are exposed in 30S subunits and inaccessible in 70S ribosomes (8-13). Some of these same studies have implicated this region in the process of subunit association (9-11).The chemical modification studies by Moazed and Noller (13,14), in which rRNA accessibilities were probed in the presence of tRNA or antibiotics, showed that the 790 loop influences several aspects of protein synthesis aside from subunit association. To further define its functional role, the universally conserved guanine at position 791 was mutated to an adenine by site-directed mutagenesis. Here we report on the structural and functional characteristics of the resulting mutant 30S subunits. MATERIALS AND METHODSMutagenesis and Expression. The single-base mutation at position 791 was constructed by oligonucleotide-directed mutagenesis in M13 as described by Zoller and Smith (15).The EcoRI-Xba I fragment from rrnB was cloned into M13mpl9 and the template was isolated from the ung-dutstrain RZ1032 to increase the frequency of mutagenesis (16). After mutagenesis M13 isolates were screened by DNA sequencing (17) and the Bgl II-Xba I fragment containing the mutation was cloned into the expression vector pKK353...

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“…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).…”

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confidence: 99%

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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Evolving Ribosome Structure and Function: rRNA and the Translation Mechanism

Cited by 4 publications

References 0 publications

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome.

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome.

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

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

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