Molecular integrity of chloroplast ribosomal ribonucleic acid

The majority of chloroplast 1.1x10(6)-mol.wt. rRNA molecules are nicked at specific points in the polynucleotide chain, the molecules being kept intact at low temperatures by their secondary structure. Conditions that break hydrogen bonds and lead to loss of secondary structure cause dissociation of the molecule.     

The synthesis of chloroplast high-molecular-weight ribosomal ribonucleic acid in spinach.

Illuminated suspensions of chloroplasts isolated from young spinach leaves show incorporation of [3H]uridine into several species of RNA. One such RNA species of Mr 2.7 x 10(6) shows sequence homology with both the chloroplast 23-S rRNA (Mr = 1.05 x 10(6)) and 16-S rRNA (Mr = 0.56 x 10(6)), as judged by DNA/RNA competition hybridization. Leaves labelled in vivo with [32P]orthophosphate in the presence of chloramphenicol accumulate labelled RNAs of Mr 1.28 x 10(6), 0.71/0.75 x 10(6) and 0.47 x 10(6). The 1.28 x 10(6)-Mr RNA shows 80.5% sequence homology with the 1.05 x 10(6)-Mr rRNA and the 0.71/0.75 x 10(6)-Mr RNA mixture shows 76% sequence homology with the 0.56 x 10(6)-Mr rRNA. We conclude that the pathway of rRNA maturation in spinach chloroplasts is similar to that of Escherichia coli.     

The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.     

Molecular integrity of plant ribosomal ribonucleic Acid

Thermal denaturation of plant ribosomal RNA followed by gel fractionation shows that although a large percentage of molecules contain breaks in the polynucleotide chain, 25S and 18S RNAs do exist as unique molecular species. Values for the rate constant of hydrolysis under routine denaturing conditions are of the order of 10⁻⁷ to 10⁻⁸ sec^{− 1} and these are shown not to be a result of ribonuclease activity. This high rate of hydrolysis and the use of insensitive fractionation procedures may account for the reported absence of a 25S rRNA molecule and its apparent conversion to a molecule similar in size to 18S RNA.     

Nucleotide sequences of chloroplast 5S ribosomal ribonucleic acid in flowering plants.

Evidence for the sequence of duckweed (Lemna minor) chloroplast 5S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The possible sequence of the chloroplast 5S rRNA from three other flowering plants was deduced by complete digestion with T1 ribonuclease and comparison of the sequences of the oligonucleotide products with homologous sequences in the duckweed 5S rRNA. This analysis indicates that the chloroplast 5S rNA species differ appreciably from their cytosol counterparts but bear a strong resemblance to one another and to the 5S rRNA species of prokaryotes. Structural features apparently common to all 5S rRNA molecules are also discussed.     

Characterization of cytoplasmic and chloroplast 5S ribosomal ribonucleic acid from broad-bean leaves

Green leaves of the broad bean (Vicia faba) contain two 5S RNA components that can be separated from each other by polyacrylamide-gel electrophoresis. The major component is located in the larger subunit of cytoplasmic ribosomes, whereas the minor component occurs in the larger subunit of chloroplast ribosomes. Their electrophoretic mobilities relative to those of Escherichia coli 5S RNA (120 nucleotides) and plant 4S RNA (78 nucleotides) suggest that they consist of 118 and 122 nucleotide residues respectively. Thermal ;melting' profiles of plant cytoplasmic and chloroplast 5S RNA species at 260nm indicate the similarity of their secondary structures, not only to each other, but also to those of E. coli and mammalian 5S RNA species. The base compositions of the two plant 5S RNA species have more in common with each other than with the corresponding molecules from either E. coli or mammalian cells.     

The secondary structure of ribosomal ribonucleic acid in solution

1. The u.v.-absorption spectrum of ribosomal RNA from rabbit reticulocytes was studied as a function of temperature at different pH values. The changes in the spectrum over the range 220-320mmu were interpreted on the basis of the assumption that the effect of denaturation and ionization are additive. The results suggest that in neutral salt solutions the secondary structure of the ribosomal RNA samples studied is due to two species of helical segments stabilized principally, if not solely, by complementary base pairs but differing in nucleotide composition: each species appears to be heterogeneous in other respects in view of the breadth of the melting ranges. 2. The number of base pairs per helical segment was estimated to be small (between 4 and 17) on the basis of the relation between melting temperature and chain length previously established by Lipsett and others for model compounds. Small fragments (about 2s) obtained by alkaline hydrolysis appeared to form the same helical segments as the intact molecule in accord with the estimated size of these segments. 3. Specific nucleotide sequences appear necessary to account for the hysteresis observed on titrating ribosomal RNA with acid or alkali within the range pH3.0-7.0 since this phenomenon was less pronounced for Escherichia coli transfer RNA and for RNA from turnip yellow-mosaic virus.     

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.     

Ribosomal ribonucleic acid and ribosomal precursor ribonucleic acid in Anacystis nidulans

The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.     

Isolation and characterization of ribosomal ribonucleic acid

1. Ribosomal RNA has been prepared by extracting tissues with a phenol–cresol mixture, and ribosomal RNA can be selectively precipitated with m-cresol. No rapidly labelled RNA was associated with this material. 2. However, if RNA and DNA are extracted with 4-aminosalicylate and phenol–cresol mixture and the nucleic acids precipitated, DNA, glycogen and s-RNA (transfer RNA) can be extracted with 3m-sodium acetate and in this case rapidly labelled RNA remains associated with the ribosomal RNA. 3. The ribosomal RNA is stable in the presence of concentrated salt solution and, although the secondary structure is lost by heating at 70° in 10mm-sodium acetate, it can be re-formed in the presence of 200mm-sodium acetate. 4. The 28s and 18s components have been separated and their base compositions determined.     

The sequence ofTetrahymena thermophila 5S ribosomal ribonucleic acid

The primary structure ofTetrahymena thermophila 5S rRNA is reported. A secondary structure model is presented which can encompass most published eukaryotic 5S rRNA sequences. Unlike other eukaryotic 5S rRNAs,Tetrahymena is found to contain the sequence-CGAAC- beginning at position 40. The presence of this segment had previously been thought to be an exclusive characteristic of eubacterial 5S rRNAs.     

Effects of different extraction procedures on the molecular characteristics of bacterial ribosomal ribonucleic acid

Ribosomal RNA has been extracted from Escherichia coli ribonucleoprotein, (RNP) in such a way as to obtain a maximal yield of 23-S particles. If RNP suspensions, with concentrations of more than 3 mg/ml, were rapidly treated with buffer-equilibrated phenol and bentonite, the relative amounts of 16-S and 23-S RNA obtained always corresponded to the relative amounts of 30-S and 50-S particles of the RNP originally present. Phenol extraction of RNP, in ionic environments varying from 0.05 M EDTA to 0.01 M MgCl₂ (in 0.01 M Tris buffer), gave 16-S and 23-S RNA in a constant ultraviolet absorbancy ratio of 1:2. As 23-S RNA is about twice the size of 16-S RNA, the numbers of each type of particle were therefore equal.Different results were observed if the concentration of the particles extracted with phenol was lowered to less than 0.1 mg/ml. When the Mg²⁺ concentration, during phenol extraction of RNP, was less than 1 mM, the ratio of 16-S to 23-S RNA produced from equal numbers of 30-S and 50-S RNP particles changed irreversibly from 1:2 to 2:1. Extraction of RNP in the presence of 0.05 M EDTA gave almost entirely 16-S RNA. No ribosomal RNA species smaller than 16-S were obtained from E. coli RNP in any of the extraction procedures.Prolonged dialysis of any of the RNA specimens against 0.05 M EDTA and bentonite did not alter the ratio of 16-S to 23-S RNA originally observed, nor was there any further dissociation of the RNA into smaller subunits. Dialysis against 8 M urea in 0.05 M EDTA also had no effect on the original ratios. Similar results were obtained after the treatment of ribosomal RNA with 0.1 M periodate at pH 6.0 or with 0.1 M hydroxylamine at pH 7.0. The passage of ribosomal RNA through sodium amberlite columns was without effect if bentonite was also present.It is concluded that both 16-S and 23-S ribosomal RNA in E. coli are molecules without readily dissociable subunit structures. The isolation of 23-S RNA is more dependent than the isolation of 16-S RNA on changes in the ionic environment during phenol extraction. This is taken to indicate the presence in 23-S RNA of labile bonds which are unduly sensitive to such changes. The observation of two different effects on the amounts of 23-S and 16-S RNA obtained from bacterial RNP, depending on the concentration of RNP during phenol extraction, is taken as evidence against ribonuclease being responsible for the dissociation of 23-S RNA to 16-S in these experiments.     

High molecular weight ribosomal ribonucleic acid from vegetative hyphae and spores of Streptomyces griseus.

High molecular weight ribosomal ribonucleic acids (rRNAs) were isolated from young vegetative cells and spores of a streptomycin non-producing Streptomyces griseus, and their electrophoretic mobility was compared to each other and to that of rRNAs of Escherichia coli K-12. The electrophoretic mobility of 23 and 16S rRNAs from vegetative cells and spores of S. griseus was identical, but the 23S rRNAs of streptomyces ribosomes migrated more slowly on polyacrylamide gel than those of E. coli ribosomes. Intact, electrophoretically homogenous rRNAs could be isolated from S. griseus (No. 45-H) only in the presence of diethyl 1 pyrocarbonate (DEP), and intact rRNAs could be obtained from spores only if DEP had been added before breaking the spores. Otherwise instead of two distinct bands, three were obtained on polyacrylamide gel.