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.     

Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl radical probing.

Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.     

The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and hydroxyl radical probing studies.

The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules. The S8 binding site on 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means. We performed in vitro iterative selection of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified. These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution. Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642). The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important. Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.     

The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography

Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.     

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.     

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA_i^Met attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA_i^Met, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A''s OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA_i^Met reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA_i^Met, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition.     

Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA.

Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.     

The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit

Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.     

Modulation of 16S rRNA function by ribosomal protein S12

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.     

Dissection of the 16S rRNA binding site for ribosomal protein S4

The ribosomal protein S4 from Escherichia coli is essential for initiation of assembly of 30S ribosomal subunits. We have undertaken the identification of specific features required in the 16S rRNA for S4 recognition by synthesizing mutants bearing deletions within a 460 nucleotide region which contains the minimum S4 binding site. We made a set of large nested deletions in a subdomain of the molecule, as well as individual deletions of nine hairpins, and used a nitrocellulose filter binding assay to calculate association constants. Some small hairpins can be eliminated with only minor effects on S4 recognition, while three hairpins scattered throughout the domain (76-90, 376-389 and 456-476) are essential for specific interaction. The loop sequence of hairpin 456-476 is important for S4 binding, and may be directly recognized by the protein. Some of the essential features are in phylogenetically variable regions; consistent with this, Mycoplasma capricolum rRNA is only weakly recognized by S4, and no specific binding to Xenopus laevis rRNA can be detected.     

Following the dynamics of changes in solvent accessibility of 16 S and 23 S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals

We have probed the structure and dynamics of ribosomal RNA in the Escherichia coli ribosome using equilibrium and time-resolved hydroxyl radical (OH) RNA footprinting to explore changes in the solvent-accessible surface of the rRNA with single-nucleotide resolution. The goal of these studies is to better understand the structural transitions that accompany association of the 30 S and 50 S subunits and to build a foundation for the quantitative analysis of ribosome structural dynamics during translation. Clear portraits of the subunit interface surfaces for 16 S and 23 S rRNA were obtained by constructing difference maps between the OH protection maps of the free subunits and that of the associated ribosome. In addition to inter-subunit contacts consistent with the crystal structure, additional OH protections are evident in regions at or near the subunit interface that reflect association-induced conformational changes. Comparison of these data with the comparable difference maps of the solvent-accessible surface of the rRNA calculated for the Thermus thermophilus X-ray crystal structures shows extensive agreement but also distinct differences. As a prelude to time-resolved OH footprinting studies, the reactivity profiles obtained using Fe(II)EDTA and X-ray generated OH were comprehensively compared. The reactivity patterns are similar except for a small number of nucleotides that have decreased reactivity to OH generated from Fe(II)EDTA compared to X-rays. These nucleotides are generally close to ribosomal proteins, which can quench diffusing radicals by virtue of side-chain oxidation. Synchrotron X-ray OH footprinting was used to monitor the kinetics of association of the 30 S and 50 S subunits. The rates individually measured for the inter-subunit contacts are comparable within experimental error. The application of this approach to the study of ribosome dynamics during the translation cycle is discussed.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.