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.     

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.     

Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

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.     

Apparent association constants for E. coli ribosomal proteins S4, S7, S8, S15, S17 and S20 binding to 16S RNA.

We have previously reported the development of a technique utilizing nitrocellulose filters, which rapidly separates ribosomal protein-ribosomal RNA complexes from unbound protein. We have used this technique to obtain binding data for the association of proteins S4, S7, S8, S15, S17, and S20 with 16S RNA. With the exception of protein S17, the association behavior for each of these proteins exhibits a single binding site with a unique binding constant. The apparent association constants have been calculated and have been found to have a range from 1.6 x 10(6) M-1 for protein S7 to 7.1 x 10(7) M-1 for protein S17. The Scatchard plot for the protein S17 binding data is biphasic, suggesting that within the RNA population two different binding sites exist, each with a different apparent association constant.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Ribosomal proteins S2, S6, S10, S14, S15 and S25 are localized on the surface of mammalian 40 S subunits and stabilize their conformation. A study with immobilized trypsin

Trypsin immobilized on collagen membranes has been used to digest accessible ribosomal proteins of rat liver 40 S subunits. Six proteins (S2, S6, S10, S14, S15 and S25) have been found to be highly exposed on the surface of 40 S particles. They appear to be in close physical contact and localized in the same region of the subunit, most likely protruding at its surface. Electric birefringence reveals that digestion of these proteins results in unfolding of subunits: the birefringence of 40 S particles becomes negative, like that of RNA, the relaxation time undergoes a 15-fold decrease and the mechanism of orientation is drastically modified.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.     

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.     

A role for proteins S3 and S14 in the 30 S ribosomal subunit

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.     

Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders.     

Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

Proteins S7, S10, S16 and S19 of the human 40S ribosomal subunit are most resistant to dissociation by salt

The protein components of human 40S ribosomal subunits were dissociated by centrifugation in gradients of sucrose and LiCl in the presence of 0.5 M KCl. The proteins that split off were analyzed by SDS-PAGE and 2D-PAGE. The order of dissociation of the proteins, depending on the salt concentration (from 0.8 M to 1.55 M), was established. The majority of the proteins started to split off simultaneously at a monovalent cation concentration of 0.8 M. Ten proteins were found to be more resistant; of these proteins S7, S10, S16, and S19 were retained most strongly and thereby may be considered to be core proteins.