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

We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.     

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.     

Probing the assembly of the 3' major domain of 16 S rRNA. Interactions involving ribosomal proteins S2, S3, S10, S13 and S14

We have used rapid probing methods to follow the changes in reactivity of residues in 16 S rRNA to chemical and enzymatic probes as ribosomal proteins S2, S3, S10, S13 and S14 are assembled into 30 S subunits. Effects observed are confined to the 3' major domain of the RNA and comprise three general classes. (1) Monospecific effects, which are attributable to a single protein. Proteins S13 and S14 each affect the reactivities of different residues which are adjacent to regions previously found protected by S19. S10 effects are located in two separate regions of the domain, the 1120/1150 stem and the 1280 loop; both of these regions are near nucleotides previously found protected by S9. Both S2 and S3 protect different nucleotides between positions 1070 and 1112. In addition, S2 protects residues in the 1160/1170 stem-loop. (2) Co-operative effects, which include residues dependent on the simultaneous presence of both proteins S2 and S3 for their reactivities to appear similar to those observed in native 30 S subunits. (3) Polyspecific effects, where proteins S3 and S2 independently afford the same protection and enhancement pattern in three distal regions of the domain: the 960 stem-loop, the 1050/1200 stem and in the upper part of the domain (nucleotides 1070 to 1190). Proteins S14 and S10 also weakly affect the reactivities of several residues in these regions. We believe that several of the protected residues of the first class are likely sites for protein-RNA contact while the third class is indicative of conformational rearrangement in the RNA during assembly. These results, in combination with the results from our previous study of proteins S7, S9 and S19, are discussed in terms of the assembly, topography and involvement in ribosomal function of the 3' major domain.     

Directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S13 using tethered Fe(II).

Directed hydroxyl radical probing was used to probe the rRNA neighborhood around protein S13 in the 30S ribosomal subunit. The unique cysteine at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) was then assembled into 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleavage of the RNA backbone in two localized regions of the 3' major domain of 16S rRNA. One region spans nt 1308-1333 and is close to a site previously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 by previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results place both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit.     

Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension

We have examined the effect of binding ribosomal protein S4 to 16 S rRNA on the susceptibility of the RNA to a variety of chemical and enzymatic probes. We have used dimethyl sulfate to probe unpaired adenines (at N-1) and cytosines (at N-3), kethoxal to probe unpaired guanines (at N-1 and N-2) and cobra venom (V1) ribonuclease as a probe of base-paired regions of 16 S rRNA. Sites of attack by the probes were identified by primer extension using synthetic oligodeoxynucleotides. Comparison of probing results for naked and S4-bound rRNA shows: Protein S4 protects a relatively compact region of the 5' domain of 16 S rRNA from chemical and enzymatic attack. This region is bounded by nucleotides 27 to 47 and 394 to 556, and has a secondary structure characterized by the junction of five helical elements. Phylogenetically conserved irregular features (bulged nucleotides, internal loops and flanking unpaired nucleotides) and helical phosphodiester bonds of four of the helices are specifically protected in the S4-RNA complex. We conclude that this is the major, and possibly sole region of contact between 16 S rRNA and S4. Many of the S4-dependent changes mimic those observed on assembly of 16 S rRNA into 30 S ribosomal subunits. Binding of S4 causes enhanced chemical reactivity coupled with protection from V1 nuclease outside the S4 junction region in the 530, 720 and 1140 loops. We interpret these results as indicative of loss of structure, and suggest that S4 binding causes disruption of adventitious pairing in these regions, possibly by stabilizing the geometry of the RNA such that these interactions are prevented from forming.     

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)     

Mapping the three-dimensional locations of ribosomal RNA and proteins.

Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory. In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy. The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA. A novel structure, the platform ring, is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site. In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory. In order to evaluate the reliability of our model for the three-dimensional distribution of 16S rRNA, we have predicted which sites of rRNA are adjacent to ribosomal proteins and compared these predictions with r-protein protection studies of others. Good correlation between the model, the locations of rRNA sites, the locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins, provides independent support for this model.     

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.     

Protein binding sites on Escherichia coli 16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9.

14C-labelled proteins from E. coli 30S ribosomal subunits were isolated by HPLC, and selected groups of these proteins were reconstituted with 32P-labelled 16S RNA. The isolated reconstituted particles were partially digested with ribonuclease A, and the RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Protein S7 alone gave no protected fragments, but S7 together with S14 and S19 protected an RNA region comprising the sequences 936-965, 972-1030, 1208-1262 and 1285-1379 of the 16S RNA. Addition of increasing amounts of protein S9 to the S7/S14/S19 particle resulted in a parallel increase in the protection of the hairpin loop between bases 1262 and 1285. The results are discussed in terms of the three-dimensional folding of 16S RNA in the 30S subunit.     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

A paradigm for local conformational control of function in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m2G966 and blocks methylation of m5C967 by their respective methyltransferases.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Nègre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid Methylation (Alan Liss, New York), pp. 1-17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C MT, while simultaneously inducing methylation by the m2G MT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G MT but not by the m5C MT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G MT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-M., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309-319). Our results show that the two ribosomal RNA MTs studied in this work are exquisitely sensitive to this small but nevertheless functionally important structural change.     

DNA-hybridization electron microscopy tertiary structure of 16 S rRNA

Seven regions of 16 S rRNA have been located on the surface of the 30 S ribosomal subunit by DNA-hybridization electron microscopy. This information has been incorporated into a model for the tertiary structure of 16 S rRNA, accounting for approximately 40% of the total 16 S rRNA. A structure labeled the platform ring is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500 to 545, and occupies a region on the exterior surface of the subunit near the elongation factor Tu binding site. Ribosomal proteins that have been mapped by immunoelectron microscopy are superimposed onto the model in order to examine possible regions of interaction. Good correlation between the model locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins provide independent support for this model.     

Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit.

The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg²⁺, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle.     

Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.