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

Abstract

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...

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

“…The data presented here have significant bearing on conflicting reports concerning the three-dimensional location of S20 in the 30S subunit+ Neutron mapping experiments have placed S20 in the head of the 30S subunit, near ribosomal proteins S3 and S10 (Capel et al+, 1987)+ In contrast, S20 has been localized near the bottom of the body of the subunit by IEM studies (Schwedler et al+, 1993)+ The IEM data locate S20 proximal to S17, consistent with conclusions drawn from footprinting experiments (Stern et al+, 1988a;Powers & Noller, 1995) and with the directed probing results presented here+ Both IEM and neutron diffraction studies localized S17 to the bottom of the body of the 30S subunit (Capel et al+, 1987;Schwedler et al+, 1993)+ The RNA regions in the 59 domain that are targeted by Fe(II)-derivatized S20 directly flank RNA elements that are cleaved by Fe(II)-S17 (G+M+ Heilek & H+F+ Noller, unpubl+ results) and Fe(II)-S15 (G+M+ Culver & H+F+ Noller, unpubl+)+ Taken together, the data are most consistent with the placement of S20 near the bottom of the body of the 30S subunit+…”

“…The binding of ribosomal protein S20 to 16S ribosomal RNA (rRNA), or fragments of 16S rRNA, has been investigated by a variety of methods (Sogin et al+, 1971;Daya-Grosjean et al+, 1974;Muto et al+, 1974;Zimmermann et al+, 1974Zimmermann et al+, , 1975Ungewickell et al+, 1975;Ehresmann et al+, 1977;Mackie & Zimmermann, 1978;Cormack & Mackie, 1991)+ Ribosomal protein S20 interacts with 16S rRNA stoichiometrically (Stöffler et al+, 1971;Muto & Zimmermann, 1978) and independently of other proteins, defining it as a primary binding protein (Mizushima & Nomura, 1970;Held et al+, 1974)+ These properties suggest that S20 plays a critical role in nucleating the assembly of the 30S subunit+ While the gene encoding S20 is not essential, strains lacking S20 have a significantly reduced growth rate (Dabbs, 1979;Götz et al+, 1989;Ryden-Aulin et al+, 1993)+ Defects in posttranscriptional modification of 16S rRNA, in vivo 30S subunit assembly, translational fidelity, and subunit association have all been identified in cells lacking S20 (Dabbs, 1979;Götz et al+, 1989;Ryden-Aulin et al+, 1993)+ Understanding the role of S20 in the ribosome is complicated not only by the multiple phenotypic effects observed in the deletion strain but also by discrepancies in the placement of S20 within the 30S subunit from different experimental approaches+ Neutron diffraction mapping has placed S20 in the head of the 30S subunit, near S3 and S10 (Capel et al+, 1987)+ In contrast, immunoelectron microscopy (IEM) has localized S20 near the bottom of the body of the 30S subunit, proximal to S17 and S15 (Schwedler et al+, 1993)+ Footprinting studies using RNA-directed probes specific to either the base or sugar moieties have identified nucleotides near positions 190, 250, 270, and 320, all elements of the 59 domain of 16S rRNA, as being some of the nucleotides protected by S20 (Stern et al+, 1988a;Powers & Noller, 1995)+ Ribosomal protein S17 also protects some of these same nucleotides from modification with base-specific chemical probes (Stern et al+, 1988a)+ This suggests that S17 and S20 are near one another in the 30S subunit+ Ribosomal protein S17 has been unambiguously positioned near the bottom of the body of the 30S subunit (Cap...…”

The Structure of Ribosomal RNA: A Three‐Dimensional Jigsaw Puzzle

“…The most serious of these concerns protein S20, which is located at the base of the head of the subunit in the neutron map. In contrast, the IEM data of Schwedler et al [98] place this protein at the lower extremity of the body of the subunit and, as Schwedler et al point out, the latter location is more compatible with the RNA-protein foot-printing data [4] or with older RNA binding site studies [99], which suggest that S20 lies close to S17; both proteins for example have foot-print sites in helix 11 of the 16s RNA (Fig. 3, bottom left).…”

“…6b), showing the IEM data of Stoffler and co-workers [66]; the IEM sites in larger letters for proteins S19 and S20 are from [97] and [98] [66], and neutron scattering [96], respectively; in both cases the view is from the interface side of the subunit. Although it is clear that for the majority of the proteins the locations determined by the two different methods are in excellent agreement, there are some exceptions.…”

A Map of Protein-rRNA Distribution in the 70 SEscherichia coli Ribosome

“…However, the SA map displays at least two protein globules at the bottom of the subunit. One of them could be S20 that has been detected (34) Fig. 1.…”