Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli.

The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new experimental information, in particular cross-linking data with functional ligands and cross-links between the 16S and 23S RNA molecules. A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model. It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed. Helix 44 is now clearly located at the interface between the 30S and 50S subunits. Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surrounding the decoding site.     

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

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.     

Altered topography of 16S RNA in the inactive form of Escherichia coli 30S ribosomal subunits.

We have studied the topography of 16S RNA in the inactive form of the 30S ribosomal subunit (Ginsburg, I., et al. (1973) J. Mol. Biol. 79, 481), using the guanine-specific reagent kethoxal. Oligonucleotides surrounding reactive guanine residues were isolated and quantitated by means of diagonal electrophoresis and sequenced. Comparison of these results with experiments on active or reactivated subunits reveals the following: (1) Most of the sites which are reactive in active 30S subunits are much more reactive (average 13-fold) in inactive subunits. Upon reactivation, these sites return to a less reactive state. Thus, a reversible increase in accessibility of specific 16S RNA sites parallels the reversible loss of protein synthesis activity of 30S subunits. (2) The number of kethoxal-reactive sites in inactive subunits is about twice that of active subunits. The nucleotide sequences and locations of the additional accessible sites in inactive subunits have been determined. (3) Sites that can be located in the 16S RNA sequence are distributed throughout the RNA chain in inactive subunits, in contrast to the clustering observed in active subunits. (4) The sites of kethoxal substitution are single stranded. Yet, of the 30 sites that can be located, 23 were predicted to be base paired in the proposed secondary structure model for 16S RNA (Ehresmann, C., et al. (1975), Nucleic Acids Res. 2, 265).     

Positions of proteins S14, S18 and S20 in the 30 S ribosomal subunit of Escherichia coli

A map of the 30 S ribosomal subunit is presented giving the positions of 15 of its 21 proteins. The components located in the map are S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S14, S15, S18 and S20.     

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli.

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.     

Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli

The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3' end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of the RNA binding site of ribosomal proteins S8 and S15.

Proteins S8 and S15 from the 30 S ribosomal subunit of Escherichia coli were bound to 16 S RNA and digested with ribonuclease A. A ribonucleoprotein complex was isolated which contained the two proteins and three noncontiguous RNA subfragments totaling 93 nucleotides, that could be unambiguously located in the 16 S RNA sequence. We present a secondary structural model for the RNA moiety of the binding site complex, in which the two smaller fragments are extensively base-paired, respectively, to the two halves of the large fragment, to form two disconnected duplexes. Each of the two duplexes is interrupted by a small internal loop. This model is supported by (i) minimum energy considerations, (ii) sites of cleavage by ribonuclease A, and (iii) modification by the single strand-specific reagent kethoxal. The effect of protein binding on the topography of the complex is reflected in the kethoxal reactivity of the RNA moiety. In the absence of the proteins, 5 guanines are modified; 4 of these, at positions 663, 732, 733, and 741, are strongly protected from kethoxal when protein S15 is bound.     

A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome

In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.     

The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation.

To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A18 mutation in this structure element. Previously, this mutation, which changes the central base pair of helix 2, C18--G917, to an A18xG917 mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested. Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits. Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control. However, the A18 mutation has a profound effect on the overall stability of the subunit. The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins. Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins. Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution. Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins. We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex. We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes.     

Comparative electron microscopic study on the location of ribosomal proteins S3 and S7 on the surface of the E. coli 30S subunit using monoclonal and conventional antibody

Summary Mice were immunised with 30S subunits from E. coli and their spleen cells were fused with myeloma cells. From this fusion two monoclonal antibodies were obtained, one of which was shown to be specific for ribosomal protein S3, the other for ribosomal protein S7. The two monoclonal antibodies formed stable complexes with intact 30S subunits and were therefore used for the three-dimensional localisation of ribosomal proteins S3 and S7 on the surface of the E. coli small subunit by immuno electron microscopy. The antibody binding sites determined with the two monoclonal antibodies were found to lie in the same area as those obtained with conventional antibodies. Both proteins S3 and S7 are located on the head of the 30S subunit, close to the one-third/two-thirds partition. Protein S3 is located just above the small lobe, whereas protein S7 is located on the side of the large lobe.     

Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide.

The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions. Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction. The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum. The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits. Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold. It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.     

The topography of ribosomal proteins on the surface of the 30S subunit of Escherichia coli

Eight ribosomal proteins, S6, S10, S11, S15, S16, S18, S19 and S21 have been localized on the surface of the 30S subunit from Escherichia coli by immuno electron microscopy. The specificity of the antibody binding sites was demonstrated by stringent absorption experiments. In addition we have reinvestigated and refined the locations of proteins S5, S13 and S14 on the ribosomal surface which had previously been localized in our laboratory (Tischendorf et al., Mol. Gen. Genet. 134, 209-223, 1974). Thus altogether 16 out of the 21 ribosomal proteins of the small subunit from E. coli have been mapped in our laboratory.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.