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.     

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.     

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.     

Neurospora crassa cytoplasmic ribosomes: cold-sensitive mutant defective in ribosomal ribonucleic acid synthesis.

Experiments were conducted to characterize further the biochemical defects of crib-1 (PJ30201), a coldsensitive mutant strain of Neurospora crassa with a defect in ribosome biosynthesis. The results are as follows. (i) The critical temperature for the expression of the mutant growth and ribosome phenotypes is in the range of 18 to 20 C. (ii) No preferential breakdown of 37S cytoplasmic ribosomal subunits synthesized by crib-1 at 25 C occurs after a shift to 10 C. (iii) Ribosomal subunits synthesized by crib-1 at 25 C function normally in in vivo protein synthesis at 10 C. (iv) Whereas wild type synthesizes both ribosomal subunits in a coordinate manner after either a temperature shift-down (25 to 10 C) of a shift-up (10 to 25 C), noncoordinate synthesis of ribosomal subunits owing to underproduction of 37S subunits occurs in the crib-1 strain immediately after a temperature shift-down. (v) After a shift from 10 to 25 C crib-1 exhibits a 12-h lag before the growth rate and the rate of synthesis of 37S subunits begin to increase significantly. (vi) At 10 C crib-1 synthesizes unequal amounts of 25S and 17S ribosomal ribonucleic acid (rRNA) molecules, resulting from a greatly reduced accumulation of stable 17S rRNA. The mutant phenotypes of crib-1 are proposed to be the result of a defect in rRNA processing.     

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).     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa.

We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA). The rDNA is found as a tandemly repeated 6.0-megadalton sequence. We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5. 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's). We have also isolated several clones containing 5S rRNA sequences. The 5S rRNA coding sequences are not found within the rDNA repeat unit. We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous.     

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.     

Characterization of Neurospora crassa polyadenylated messenger ribonucleic acid: structure of the 5'-terminus.

Polyadenylated (poly(A)⁺) mRNA from Neurospora crassa was isolated by affinity chromatography on poly(U) Sepharose and its structure was examined. Two 5′-terminal ·cap’ structures, m⁷G(5′)ppp(5′)Ap and m⁷G(5′)ppp(5′)Gp, occurring in a relative distribution of 75 and 25% were found. No evidence was obtained for 2′-O-methylation in a nucleotide adjacent to the 5′-terminal cap.     

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.     

Levels and rates of synthesis of ribosomal ribonucleic acid, transfer ribonucleic acid, and protein in Neurospora crassa in different steady states of growth.

The levels of ribosomes, tRNA molecules, and total protein per genome in Neurospora mycelia have been determined in eight different conditions of exponential growth. By increasing the rate of growth the number of ribosomes per genome increases dramatically while the level of total protein remains almost unchanged and the level of tRNA increases only slightly. The rates of synthesis of each of the macromolecules have been estimated. Increasing the rate of growth (mu) up to 0.5, the ratio between the rates of synthesis of tRNA and rRNA decreases reaching a constant value. The equations that best describe the dependence of the rate of synthesis of the macromolecules on the rate of growth (mu) have been determined. The rate of rRNA synthesis (rr), expressed as nucleotides polymerized, min- minus 1 per genome, is given by the equation: rr equals 6.51 times 10-7 mu-2-19. The rate of protein synthesis (rp), expressed as amino acids polymerized, min- minus 1 per genome is given by the following relationship: rp equals -1.43 times 10-7 + 3.43 times 10-8 mu. The equation describing the tRNA synthesis (rt) expressed as nucleotides, min- minus 1 per genome is rt equals 6.45 times 10-5 times exp 2.30 mu; however, more accurate determinations appear to be required for a firmer assignment of this latter equation. The significance of these equations for the studies on the regulation of rRNA and protein synthesis is discussed. For instance the rate of rRNA synthesis may set the limit for the maximal growth rate attainable by a cell, as the maximal rate of rRNA synthesis that may take place in a given cell is limited by the degree of redundancy of the rRNA genes.     

Acidic ribosomal proteins of Neurospora crassa

Neurospora crassa acidic ribosomal proteins from the high salt-ethanol extract of 80 S ribosomes have been fractionated by DEAE-cellulose chromatography. Six acidic ribosomal proteins were purified. All resemble Escherichia coli L7 and L12 in amino acid composition and molecular weight but each has a slightly different net charge at pH 3.2. Four have an apparent molecular weight of approx. 14 000, and two have a molecular weight of approx. 14 800. The amino acid compositions and circular dichroism (CD) spectra of the purified Neuropsora proteins are identical for the four 14 kDa proteins, but clearly distinguishable from the two 14.8 kDa proteins. The latter are also identical in amino acid composition and CD spectra. This suggests that there are two Neurospora acidic, or 'A', proteins, one of which exists in four microheterogeneous forms and the other exists in two forms.