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.     

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.     

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.     

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.     

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).     

Homologies in eukaryotic 5.8S ribosomal RNA.

The primary nucleotide sequence of Novikoff hepatoma ascites cell 5.8S rRNA (also known as 5.5 or 7S RNA) has been determined to be:

This sequence is 75% homologous with the primary nucleotide sequence of yeast 5.8S rRNA and 100% homologous with oligonucleotide marker fragments from HeLa cell RNA. In constrast, only limited homology is evident with oligonucleotides from 5.8S RNA of several flowering plants and many of the characteristic fragments differ.     

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.     

Identification of fibronectin fragments that bind to carboxy-group-modified proteins.

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.     

Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin

Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin (K_d ≈ 2 nm), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μm riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin (K_m ≈ 12 μm), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate (K₃ ≈ 5 nm) but less avidly complexes riboflavin (K_d ≈ 0.6 μm), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin.     

Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.