Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Isolation and characterization of membrane-bound ribosomes from nerve cell bodies of immature rat brain-cortex.

Free and membrane-bound ribosomes were isolated from neuronal perikarya of the immature rat brain-cortex. The two topographic forms of ribosomes were essentially free of contaminating organelles as shown by RNA, protein and marker enzyme analysis. Membrane-bound ribosomes amount to about a quarter of the total ribosomal population in neuronal perikarya. Both forms of ribosomes efficiently carried out cell-free protein synthesis but the membrane-bound fraction was more active than the free ribosomes.     

Distribution of membrane-bound and free ribosomes in growing yeast.

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. the relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly (U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317-328).     

Mechanism of ribosomal subunit association: oligodeoxynucleotides as probes.

From the kethoxal treatment data [Herr, W.; Chapman, N.M.; Noller, H.F. (1979) J. Mol. Biol. 130, 433-439] some regions of ribosomal RNAs are thought to be responsible for the association of 30S and 50S ribosomes of E. coli to form 70S ribosomes. In order to test this possibility about a dozen oligodeoxynucleotides complementary to the suspected regions of rRNAs were synthesised. Their association with ribosomes and naked rRNAs was tested by the gel filtration technique. In order to check the effects on the ribosomal subunit association or rRNA association either intact 30S and 50S ribosomes or naked 16S and 23S rRNAs were preincubated with the individual oligodeoxynucleotide and its effect was checked by density gradient centrifugation followed by UV absorbance monitoring. Some oligodeoxynucleotides interfered with either subunit association or 16S RNA and 23S RNA association, some with both. These data clearly indicate that RNA-RNA interaction plays the major role in ribosomal subunit association.     

Absence of ribosomes in Capsicum chromoplasts

Ribosome development was followed by electron microscopy and gel electrophoresis of ribosomal (r)RNAs in the plastids of fully expanded fruits of Capsicum annuum L. during ripening. Chloroplasts from young Capsicum leaves were used as a structural and electrophoretic standard. Four stages were distinguished on the basis of colour changes during fruit ripening. Chloroplasts of the green fruit had a lower content of 16S and 23S rRNAs than leaf chloroplasts. They contained only a few ribosomes, some more discrete ribosomal particles, and the contrast of ribosomal structures was faint. From the outset of ripening, most of the ribosomal structures in the plastid stroma disappeared. A continuous decrease in plastid rRNAs occurred during ripening. Fully differentiated chromoplasts of the red fruit did not contain rRNAs or ribosomes. Throughout plastid development, DNA nucleoids were evident and there was only a small decrease in the DNA peak on electrophoretograms. The loss of ribosomes during the chloroplast-to-chromoplast conversion in Capsicum fruit is discussed in relation to the variations in pigments and enzymic systems in both plastid types.Abbreviations Developmental stages of leaves and fruits: A four-week-old green leaf - B green fruit - C brownish fruit - D orange fruit - E red fruit - ptRNA, DNA plastid RNA - DNA; rRNA ribosomal RNA     

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence

The quantity of RNA in the ribosomal fraction of the first leaf of cucumber (Cucumis sativus) increases during growth, reaches a maximum before the final fresh weight is attained, and then decreases. The main changes are in the free ribosome fraction, the quantity of membrane-bound ribosomes remaining about constant. Few 65.5S chloroplast ribosomes are present in small leaves; however, they increase in quantity rapidly during growth and form about half of the ribosomes present in the mature fully green leaf. The cytoplasmic ribosomes have a sedimentation coefficient of 77.6S. Ribonuclease-sensitive polysomes were present in leaves of all ages except possibly the very oldest. The proportion of ribosomes in polysome form decreases during growth and then remains roughly constant during senescence. Following maturation of the leaf, the rate of incorporation of ³²P into ribosomal-fraction RNA begins to decline. This decline could account for the loss of ribosomes during the early stages of senescence. The possibility that leaf ribonuclease might be responsible for the final, more rapid loss of RNA, is discussed.     

Unequal accumulation of 26S and 17S RNAs in ribosomes during spore germination in Dictyostelium discoideum

Ribosome synthesis was studied in spores at the swelling stage and compared with freshly emerged and logarithmically growing vegetative amoebae. During the swelling stage of spore germination, ribosome synthesis was abnormal. Newly made ribosomes accumulated unequal amounts of 26S and 17S rRNAs. The stoichiometric ratio 26S:17S was 0.5 in swelling spores, compared with 0.9 in amoebae. The relative level of pre-rRNA persisting in the nucleus was apparently 2- to 3-fold higher in swelling spores than in amoebae. All of the known ribosomal proteins, except for a few, were made during the swelling stage and were associated with the newly made ribosomes in expected amounts. Analysis of the 2'-O-methyl ribose content in the newly made rRNAs suggest that methylation was defective in swelling spores. Compared with growing amoebae, the methyl content was 30 and 64% less in 26S and 17S RNAs from the swelling stage, respectively. It is suggested that undermethylation could be partly responsible for the differential accumulation of newly made 26S and 17S RNAs during the early stages of spore germination in Dictyostelium discoideum.     

In vitro incorporation of eubacterial, archaebacterial and eukaryotic 5S rRNAs into large ribosomal subunits of Bacillus stearothermophilus.

Bacillus stearothermophilus large ribosomal subunits were reconstituted in the presence of 5S rRNAs from different origins and tested for their biological activities. The results obtained have shown that eubacterial and archaebacterial 5S rRNAs can easily substitute for B. stearothermophilus 5S rRNA in the reconstitution, while eukaryotic 5S rRNAs yield ribosomal subunits with reduced biological activities. From our results we propose an interaction between nucleotides 42-47 of 5S rRNA and nucleotides 2603-2608 of 23S rRNA during the assembly of the 50S ribosomal subunit. Other experiments with eukaryotic 5.8S rRNAs reveal, if at all, a very low incorporation of these RNA species into the reconstituted ribosomes.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Prediction of the recognition sites on 16S and 23S rRNAs from E. coli for the formation of 16S-23S rRNA complex

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E. coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Nuclease S1 mapping of 16S ribosomal RNA in ribosomes

Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes. The quantitative disposition of these sequences within ribosomes is not known. Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes. Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired. Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA. The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1. In deproteinated E. coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs. However, the quantitative disposition of the sequences in 70S ribosomes varies with each position. In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.