Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.     

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.     

THE NATURE AND PROCESSING OF RIBOSOMAL RIBONUCLEIC ACID IN A DINOFLAGELLATE

Certain features of the dinoflagellate nucleus suggest that it represents a primitive form of eukaryotic nucleus. For this reason, it was of interest to characterize dinoflagellate ribosomal RNA (rRNA) and its mode of synthesis to determine if it also deviated from typical eukaryotic patterns. Gyrodinium cohnii was chosen for this examination. Gyrodinium ribosomal RNA species are 16 and 25s as judged by their sedimentation velocities in isokinetic sucrose gradients. These values are typical of higher plants. In addition, the RNA cosedimented precisely with rRNA from the ciliate Tetrahymena. Nucleotide ratio analyses revealed a GMP + CMP content of 46% for both species of rRNA. The kinetics of incorporation of a radioactive precursor into ribosomal RNA have also been studied, and it seems likely that the maturation of rRNA starts with the synthesis of a 38s molecule. This serves as precursor to the 16s species, and, after a 27s intermediate, the 25s ribosomal component. The process is similar to that in other eukaryotes. The structure of the nucleolus has also been examined, and is seen to be typically eukaryotic.     

Comparative Study of Ribosomal Ribonucleic Acid Cistrons in Enterobacteria and Myxobacteria

Deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybrids are formed by Escherichia coli 16S or 23S ribosomal RNA or pulse-labeled RNA with the DNA of various species of the Enterobacteriaceae. The relative extent of hybrid formation is always greater for ribosomal RNA. These DNA-RNA hybrids have been further characterized by their stability to increasing temperature, and, in every case, the stability of pulse-labeled RNA hybrids was lower than that of the corresponding ribosomal RNA hybrids, although 16S and 23S ribosomal RNA hybrids had very similar stabilities. Therefore, ribosomal RNA showed a greater degree of apparent conservation in base sequence than pulse-labeled or messenger RNA both in the extent of cross-reaction and in the stability of hybrid structures. Similar results were obtained with Myxococcus xanthus RNA. Since in this case the base composition of the pulse-labeled or messenger RNA is richer in guanine plus cytosine than ribosomal RNA, the higher cross-reaction of ribosomal RNA is more readily attributable to conservation of base sequence in these cistrons than to its base composition. Thus, the base sequence of ribosomal RNA cistrons of bacilli, enteric bacteria, and myxobacteria is conserved relative to those of the rest of the genomes. This conservation is, however, not absolute since the stability of heterologous ribosomal RNA hybrids is always lower than that of homologous hybrids.     

Phylogenetic Origin of the Chloroplast*†

SYNOPSIS. The 16S ribosomal RNA of the chloroplast of Euglena gracilis strain Z has been characterized in terms of its 2-dimensional electrophoretic “fingerprint” (T1 ribonuclease). Over 100 spots were resolved on the “fingerprint” and each spot was characterized as to which RNA oligonucleotide fragment(s) it contained. When compared to similar analyses of prokaryotic 16S rRNAs and eukaryotic cytoplasmic 18S rRNAs, the chloroplast 16S rRNA was a typically prokaryotic RNA, but bore little if any relationship to eukaryotic 18S rRNAs. Therefore, the cistrons for chloroplast 16S rRNA are related to the equivalent prokaryotic cistrons, but, apparently, are not related to the equivalent eukaryotic cistrons. Among the organisms available for comparison, the Euglena chloroplast 16S rRNA appears most closely related to the 16S rRNA of the eukaryote, Porphyridium cruentum (a red alga), and at least distantly related to the 16S rRNAs of the blue-green algae and perhaps also to the bacilli.     

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.     

On the interpretation of small-angle x-ray solution scattering from spherical viruses.

The intermediates in the ribosome assembly in exponentially growing Escherichia coli have been identified by centrifuging a crude lysate, pulse-labeled with a radioactive RNA base, through a sucrose gradient and analyzing for precursor rRNA in the gradient fractions by gel electrophoresis. The major intermediate in the assembly of the 50 S subunit cosediments with the mature subunit, whereas two minor precursor species sediment between the 30 S and 50 S peaks. The assembly of the 30 S subunit proceeds via a minor intermediate sedimenting slightly behind the mature subunit and a major precursor particle that cosediments with the mature 30 S subunit.The fraction of the rRNA contained in these precursor particles was determined by direct determination of the amount of rRNA in the precursor particles, and from the labeling kinetics of their rRNA. The direct estimation indicated that about 2% of the total 23 S type RNA, and 3 to 5% of the total 16 S type RNA is harboured in precursor particles. In the kinetic experiments the specific activity of the nucleoside triphosphates and of the different ribosomal particles was followed after addition of a radioactive RNA precursor to the growth medium. The results were compared with a digital simulation of the flow of isotopes through the assembly pathways. This method indicated that approximately 2% of the total 23 S type RNA, as well as 2% of the total 16 S type RNA, is contained in the precursor particles.     

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′-end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70-75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0.23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0.23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RC(rel) mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RC(rel) strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.     

A ribosomal RNA operon from Pseudomonas stutzeri Zobell

A ribosomal RNA operon from the marine bacterium, Pseudomonas stutzeri Zobell, was cloned and characterized by Southern hybridization and sequence analysis. The 16S rRNA, 23S rRNA, 5S rRNA and 2 tRNA genes (alanine and isoleucine) were identified by homology with sequences in GenBank. The rRNA gene exhibited typical eubacterial organization (16S-tRNAs-23S-5S). A putative ribosomal promoter and anti-terminator regions were also identified and described. Significant differences in spacing of the anti-terminator regulatory elements were observed between P. stutzeri Zobell and Escherichia coli.     

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA, Sauromatum but not Olisthodiseus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [³H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.     

Precursor of 5S Ribosomal Ribonucleic Acid in the Blue-Green Alga Anacystis nidulans

The maturation of 5S ribosomal ribonucleic acid (rRNA) in the obligately photoautotrophic unicellular blue-green alga Anacystis nidulans has been studied by using polyacrylamide gel electrophoresis and T1 ribonuclease oligonucleotide analysis. A. nidulans mature 5S rRNA (m5) is of approximately the same molecular weight as the 5S rRNA of Escherichia coli, and is derived by cleavage of a precursor (p5) containing a few (three to six) additional nucleotides. Some of these additional nucleotides occur at the 5' end of the precursor molecule; others may occur at the 3' end. Kinetic experiments indicate that precursors of mature 5S rRNA larger than p5 either do not exist or are very transient in A. nidulans. These results are discussed in relation to those obtained with other prokaryotes.