Processing pathway of Escherichia coli 16S precursor rRNA.

Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA.     

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.     

Ribosomal Ribonucleic Acid Synthesis and Maturation in the Blue-Green Alga Anacystis nidulans

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.     

Quantitative determination of human bone marrow proliferation kinetics during three culture days]

In order to obtaon of human bone marrow cells, fresh bioptic material was homogenized and the cell suspensions were incubated for 72 hs in a fluid medium. After 24, 48 and 72 hs of incubation the total cell number of the culture was determined. At the same time differential counts of stained smears were performed. Both, erythrocytopoiesis and granulocytopoiesis showed regeneration, maturation, and an absolute increase of the number of precursors and of mature cells. The quantitative data obtained in vitro during 24 hs correspond with our data of kinetics obtained by observed mitotic duration and cell differential countings in vivo. However, after a longer cultivation time we found a diminution of divisible precursors, and an increase of mature erythroblasts as well as an excessibe survival of the PMNs.     

The molecular weights of yeast ribosomal precursor RNAs

The molecular weights of the predominant rRNA precursors as well as those of 26-S and 17-S mature rRNA from Saccharomyces carlsbergensis were determined by polyacrylamide gel electrophoresis in the presence of formamide. Mature 26-S + 5.8-S rRNA was found to have a molecular weight of 1.24 X 10(6) while their immediate precursor, 29-S RNA, had a molecular weight of 1.52 X 10(6). Values of 0.70 X 10(6) and 0.82 X 10(6) were obtained for the molecular weights of mature 17-S rRNA and its 18-S precursor. Finally the 37-S precursor, common to both 29-S and 18-S RNA, was found to have a molecular weight of 2.80 X 10(6). Each precursor rRNA, therefore, contains extra sequences not found at the next stage of maturation.     

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.     

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.     

Ordered processing of Escherichia coli 23S rRNA in vitro.

In an RNase III-deficient strain of E. coli 23S pre-rRNA accumulates unprocessed in 50S ribosomes and in polysomes. These ribosomes provide a substrate for the analysis of rRNA maturation in vitro. S1 nuclease protection analysis of the products obtained in in vitro processing reactions demonstrates that 23S rRNA processing is ordered. The double stranded stem of 23S rRNA is cleaved by RNase III in vitro to two intermediate RNAs at the 5' end and one at the 3' end. Mature termini are then produced by other enzyme(s) in a soluble protein fraction from wild-type cells. The nature of the reaction at the 5' end is not clear, but the reaction at the 3' end is exonucleolytic, producing three heterogeneous mature termini. The two reactions are coordinated; 3' end maturation progresses concurrently with cleavages at the 5' end. Two results suggest a possible link between final maturation and translation: in vitro, mature termini are formed efficiently in the presence of additives required for protein synthesis; and all the processing intermediates detected from in vitro reactions are also found in polysomes from wild-type cells.     

Comparative study of RNA metabolism in freshly isolated protoplasts and callus cultures of Parthenocissus tricuspidata crown gall

RNA metabolism was compared in protoplasts and cells of Parthenocissus tricuspidata crown gall callus. A rapid increase in the permeability to precursors during the first three hours following the termination of protoplast isolation was observed. Consequently, RNA synthesis occurred at a higher rate in protoplasts than in whole cells. On the other hand, protoplasts and callus tissues showed similar kinetics of incorporation of precursors into mature rRNA's. Pulse-chase experiments showed the maturation rate to be nearly the same in both cases.     

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.     

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.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Separation of Bacterial Ribosomal Ribonucleic Acid from Its Macromolecular Precursors by Polyacrylamide Gel Electrophoresis

Electrophoresis on polyacrylamide gels was found to be a powerful technique for separating the mature from the precursor forms of bacterial ribosomal nucleic acid (rRNA). The separation of the 16S rRNA from its precursor was, for all practical purposes, complete; that of the 23S rRNA from its precursor was detectable but incomplete. When mature and precursor rRNA preparations were heated to randomize secondary structure, etc., and then cooled, it was found that electrophoretic mobility differences between mature forms of rRNA and their precursors persisted. This, in conjunction with the rather large electrophoretic mobility differences between mature and precursor forms, can be taken as strong evidence for a molecular weight difference between mature rRNA and its precursor forms of RNA. With the 16S rRNA, this difference could be as large as 130,000 daltons.     

The ordered processing of intervening sequences in 23S rRNA of Rhodobacter sphaeroides requires RNase J

The essential processing of ribosomal rRNA precursors requires concerted and sequential cleavages by different endo- and exoribonucleases. Despite long lasting investigations of these processes the exact order of steps remained elusive. Many bacteria perform additional rRNA processing steps by removing intervening sequences within the 23S rRNA. This leads to disintegration of the 23S rRNA and discontinuously assembled fragments within the ribosomes. The maturation of these fragments also requires successive cleavage events by different RNases. Our study reveals that the 5'-to-3' exoribonuclease RNase J is responsible for the final 5'-end maturation of all three 23S rRNA fragments in the α-proteobacterium Rhodobacter sphaeroides. Additionally the results show that 5'- and 3'-processing steps are closely coupled: mature 5'-ends are a strict prerequisite for the final 3'-trimming of the 23S rRNA fragments.     

Maturation of black spruce somatic embryos: Sucrose hydrolysis and resulting osmotic pressure of the medium

The physiological and osmotic roles of sucrose during black spruce (Picea mariana (Mill.) B.S.P.) embryo maturation were investigated. The results showed that when both sucrose and mannitol were present in the medium, the optimum sucrose concentration varied between 4% and 6%. From these data, mannitol does not apparently replace sucrose during the maturation of somatic embryos and therefore it might not be a suitable osmoticum. For the media supplemented with 4% to 12% sucrose and various concentrations of mannitol, the osmotic pressure of the medium rose during maturation, particularly for the highest sucrose concentrations (7% to 12%). Medium containing 3% each of fructose and glucose produced fewer mature embryos compared to the medium with 6% sucrose. An increment in the osmotic potential was observed in medium with 6% sucrose in contrast to that containing 3% each of fructose and glucose. Sugar analysis revealed that the sucrose hydrolysis in the medium was detectable within 1 week of incubation and continued throughout the maturation period. Moreover, no significant uptake of the sugars was detected, since the total amount of fructose, glucose and sucrose remained constant. Our results indicate that the action of sucrose on embryo maturation is mostly achieved through an osmotic control.     

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.