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.     

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.     

Neutron-scattering studies of the ribosome of Escherichia coli: a provisional map of the locations of proteins S3, S4, S5, S7, S8 and S9 in the 30 S subunit.

Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed.     

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 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.     

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.     

Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26

The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His)(6)-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.     

Synthesis and anthelmintic activity of cyclohexadepsipeptides with (S,S,S,R,S,R)-configuration

The (S,S,S,R,S,R)-configurated cyclohexadepsipeptides (CHDPs) represent novel enniatin derivatives with strong in vivo activity against the parasitic nematode Haemonchus contortus Rudolphi in sheep. 2D NMR spectroscopic analysis revealed for the major conformation the asymmetric conformer, containing a cis-amide bond between C(alpha) protons of neighbouring 2-hydroxy-(S)-carboxylic acid and N-methyl-(S)-amino acid. The absolute configuration of the novel CHDPs was determined by X-ray crystallography. A correlation between the major conformer and its anthelmintic activity was found. Here, we report on a simple total synthetic pathway for this particular type of CHDPs.     

The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.     

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.     

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.     

Sequences of the cDNAs and genomic DNAs encoding the S1, S7, S8, and Sf alleles from almond, Prunus dulcis

Partial genomic and cDNA sequences of the RNase alleles S1, S7, S8 and Sf were obtained from Prunus dulcis cvs ’Ne Plus Ultra’, ’Ferragnes’ and ’Nonpareil’ 15–1, and IRTA Selection 12–2. Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from ’Ferragnes’, and the S7 and S8 alleles from ’Nonpareil’ 15–1, matched those reported in the literature for the alleles Sb, Sc and Sd respectively. The sequences of the S1, S7, S8 and Sf alleles found in genomic DNA contained introns of 562, 1,530, 2,208 and 689 bp respectively. The exon/intron splice junction sites of all alleles followed the GT/AG consensus sequence rule, and the sequences were found to be highly conserved. Received: 18 October 2000 / Accepted: 8 March 2001     

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.     

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)     

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle.

Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.     

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.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S7, S9, S10, S11, S17, S18 and S21 by treatment with bis-(2-chloroethyl)-methylamine.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with bis-(2-chloroethyl)-methylamine. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: proteins S4 and S9 are cross-linked to the 16S RNA at positions 413 and 954, respectively. Proteins S11 and S21 are both cross-linked to the RNA within an oligonucleotide encompassing positions 693-697, and proteins S17, S10, S3 and S7 are cross-linked within oligonucleotides encompassing positions 278-280, 1139-1144, 1155-1158, and 1531-1542, respectively. A cross-link to protein S18 was found by a process of elimination to lie between positions 845 and 851.     

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.