Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.     

The structure of rat 28S ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene.

The nucleotide sequence of a rat 28S rRNA gene was determined. The 28S rRNA encoded in the gene contains 4718 nucleotides and the molecular weight estimated from the sequence is 1.53 x 10(6). The guanine and cytosine content is 67%. The sequence of rat 28S rRNA diverges appreciably from that of Saccharomyces carlsbergensis 26S rRNA (about 50% identity), but more closely approximates that of Xenopus laevis 28S rRNA (about 75% identity). Rat 28S rRNA is larger than the analogous nucleic acids from yeast (3393 nucleotides) and X, laevis (4110 nucleotides) ribosomes. The additional bases are inserted in specific regions and tend to be rich in guanine and cytosine. 5.8S rRNA can interact with 28S rRNA by extensive hydrogen bonding at two sites near the 5' end of the latter.     

Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column.     

Maturation pathway for Novikoff ascites hepatoma 5.8 S ribosomal ribonucleic acid. Evidence for its presence in 32 S nuclear ribonucleic acid.

Evidence that 32 S nRNA contains 5.8 S rRNA was provided by studies on specific oligonucleotide sequences of these RNA species. Purified 32P-labeled 5.8 and 28 S rRNA and 32 S RNA were digested with T-1 ribonuclease, and the products were fractionated according to chain length by chromatography on DEAE-Sephadex A-25 at neutral pH. The oligonucleotides in Peak 8 were treated with alkaline phosphatase and the products were separated by two-dimensional electrophoresis on cellulose acetate at pH 3.5 and DEAE-paper in 7% formic acid. Seven unique oligonucleotide markers for 5.8 S rRNA including the methylated octanucleotide A-A-U-U-Gm-G-A-Gp were present in 32 S RNA but were not found in 28 S rRNA, indicating that 5.8 S rRNA is directly derived from the 32 S nucleolar precursor. These studies confirm a maturation pathway for rRNA species in which 32 S nucleolar RNA is a precursor of 5.8 S rRNA as well as 28 S rRNA.     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

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.     

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)     

Structural analyses of mammalian ribosomal ribonucleic acid and its precursors. Nucleotide sequence of ribosomal 5.8 S ribonucleic acid.

The nucleotide sequence of ribosomal 5.8 S RNA (also known as 7 S or 5.5 S rRNA) from Novikoff hepatoma ascites cells has been determined to be (see article). Estimations of the secondary structure based upon maximized base pairing and the fragments of partial ribonuclease digestion indicate that there may be five base-paired regions in the molecule, three forming a folding of the termini and two forming secondary hairpin loops. The sequence of Novikoff hepatoma 5.8 S rRNA is about 75% homologous with that of yeast 5.8 S rRNA (Rubin, G.M. (1973) J. Biol. Chem. 248, 3860-3875) and similar models for secondary structure are proposed. Both models contain a very stable G-C rich hairpin loop (residues 116 to 138), a less stable A-U-rich hairpin loop (residues 64 to 91) and two symmetrical bulges (residues 15 to 25 and 40 to 44).     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

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.     

The presence of a high-molecular-weight (guanine-plus-cytosine)-rich segment at the 3'' end of rabbit 28S ribosomal ribonucleic acid.

The 3' hydroxyl end of 28S L-rRNA (major RNA species of the larger subribosomal particle) was labelled by coupling its 2-hydroxy-3-naphthoic acid hydrazine with diazotized [3H]aniline. The RNA was hydrolysed partially with ribonuclease T1 and fractionated on Sephadex G-200. The results show that a highly structured segment with 78% G+C content and a number-average molecular weight of at least 1.0x10(5)-1.8x10(5) is located at the 3' hydroxyl end of the 28S rRNA molecule.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.     

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A 12 S precursor to 5.8 S rRNA associated with rat liver nucleolar 28 S rRNA

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S RNA from mouse (Mr, 0.36 X 10(6)) is larger than the one from rat (Mr, 0.32 X 10(6). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.     

Studies on the 3′-terminal sequences of the large ribosomal ribonucleic acid of different eukaryotes and those associated with `hidden'' breaks in heat-dissociable insect 26S ribonucleic acid

The 3'-terminal sequences associated with the large rRNA complex from a range of eukaryotes were determined after pancreatic or T(1)-ribonuclease digestion of RNA terminally labelled with [(3)H]isoniazid. In all higher eukaryotes examined except Drosophila melanogaster, the 3'-terminal sequences Y-G-U(OH) and G-C-U(OH) were demonstrated for the large RNA component(s) and for 6S RNA respectively. The 3'-terminal sequence of Saccharomyces cerevisiae 26S RNA was Y-G-U(OH) and that of 6S RNA Y-A-U-U-U(OH). Three 3'-terminal sequences were found in equimolar amounts in the heat-dissociable 26S rRNA characteristic of insect ribosomes. These were Y-G-U-G-U(OH), Y-C-G-U(OH) and G-C-U(OH) for cultured Antheraea eucalypti cells, Y-G-U(OH), Y-G-U(OH) and G-C-U(OH) for Galleria mellonella larvae and Y-C-G-A(OH), Y-G-U-A(OH) and G-Y-U-G(OH) for Drosophila melanogaster flies. Thus the introduction of the central scission in insect 26S rRNA results in the generation of a unique 3'-terminus and does not arise from random cleavage of the polynucleotide chain.