45S rRNA Precursor Research Articles

DNA sequences of promoter regions for rRNA operons rrnE and rrnA in E. coli

The nucleotide sequences have been determined for the promoter regions of two ribosomal RNA operons, rrnA and rrnE, in E. coli. The sequences cover the two in vitro transcription start sites identified for each operon (Gilbert, de Boer and Nomura, 1979). The first two start sites are 283 and 291 bp preceding the mature 16S rRNA (m16S rRNA) coding regions for rrnE and rrnA, respectively; the second start sites are 174 and 174 ± 1 bp preceding the m16S rRNA coding regions for rrnE and rrnA, respectively. Each of these start sites has an identifiable “Pribnow box” sequence 6–7 bp upstream from the start site. The nucleotide sequences of the two operons have nearly complete homology from the m16S rRNA coding regions to positions 145 bp upstream from those regions, and at the regions surrounding the Pribnow boxes preceding the first start sites. The DNA sequences indicate that the RNAs transcribed from the first start sites of rrnE and rrnA are quite different in their first 150 nucleotides. These heterogeneous regions, however, precede the RNAse III cleavage sites (deduced previously by Young and Steitz, 1978), and the “precursor 16S rRNA” molecules are largely homogeneous. The nucleotide sequences of the promoter regions of the two rRNA operons are also compared with those of rrnD and rrnX, determined by Young and Steitz (1979), and some common features are discussed.

Processing of the 5' end of Escherichia coli 16S ribosomal RNA.

We have isolated and partially characterized an endonuclease involved in processing the 5' end of 16S rRNA of Escherichia coli. A mutant strain that is deficient in this enzyme accumulates a new precursor of 16S rRNA, named 16.3S rRNA. This rRNA has the 3' end of mature 16S rRNA but is about 60 nucleotides longer at the 5' end. In vitro, the enzyme preparation cleaves an RNA fragment of about 60 nucleotides from the 5' end of 16.3S rRNA in 30S ribosomal subunits, yielding the mature 5' end of 16S rRNA. In the mutant strain the 16.3S rRNA is associated with a full complement of 21 ribosomal proteins in 30S subunits. These particles, which comprise 50% of the total 30S subunits, are present on polyribosomes.

Transcriptional properties of nucleoli isolated from Tetrahymena.

Nucleoli can be isolated from Tetrahymena in a yield of 30-60%. The isolated nucleoli contain rDNA (at least 90% pure) and have a protein to DNA ratio of 30:1. The endogenous RNA-polymerase activity of the r-chromatin has the following properties: (i) The in vitro transcript has a maximal size identical to the in vivo 35S rRNA precursor, demonstrating correct termination on the gene, (ii) 79% of the in vitro transcript is complementary to cDNA of 17S and 25S rRNA which is close to the theoretical maximum for the 35S rRNA precursor, (iii) the elongation rate of the endogenous RNA-polymerase molecules is 9-12 nucleotides/sec, (iv) an average of 4-16 active RNA polymerases are associated with each rDNA molecule depending upon the preparation.

Distribution of messenger RNA-coding sequences in fractionated chromatin

Chromatin from Friend erythroleukemia cells has been fractionated according to the DNAase II/ Mg ++ solubility method of Gottesfeld et al. (1974). Chromatin was separated into nuclease-sensitive and -resistant fractions after mild DNAase II digestion; the enzyme-solubilized material was further fractionated into Mg ++-soluble and -insoluble fractions. The Mg ++-soluble fraction of Friend cell chromatin represents 15–18% of total chromosomal DNA. The distribution of transcribed and nontranscribed DNA sequences in these chromatin fractions has been determined. When grown under standard conditions of cell culture, Friend cells contain only low levels of globin mRNA (about 130 copies per cell cytoplasm). Upon stimulation with 1.5% dimethylsulfoxide, these cells produce 10-fold higher amounts of cytoplasmic globin message. While no enrichment for globin nucleotide sequences is found in the Mg ++-soluble fraction of nonstimulated cells, globin sequences are significantly more abundant in the Mg ++-soluble fraction of dimethylsulfoxide-treated cells. DNA sequences complementary to poly(A)-containing cytoplasmic RNA are also preferentially localized in the Mg ++-soluble fraction of Friend cell chromatin. Upon DMSO induction, transcription of 45S rRNA precursor is inhibited ( Sherton and Kabat, 1976). We find that DNA sequences coding for rRNA are 4–5 fold more abundant in the Mg ++-soluble fraction of uninduced cells than in the insoluble chromatin from these cells; no such difference, however, is found between the chromatin fractions obtained from DMSO-induced cells. Nontranscribed highly repetitive DNA sequences are concentrated in the inactive chromatin fraction. These data support the hypothesis that the Mg ++-soluble fraction of isolated chromatin corresponds to the portion of the genome which is transcriptionally active in vivo.

Spacer transfer RNAs in ribosomal RNA transcripts of E. coli: Processing of 30S ribosomal RNA in vitro

At least three different transfer RNAs are produced by in vitro processing of 30S ribosomal RNA which accumulates in RNAase III − strains of E. coli. Two of these tRNAs, tRNA 2 Glu and tRNA 1 He, have previously been shown to be “spacer tRNAs”—that is, genes for their synthesis are located in rRNA transcription units between the cistrons for 16S and 23S rRNAs (Lund et al., 1976). The third tRNA whose sequences are contained in 30S rRNA is tRNA 1B Ala. In addition to the tRNAs, 5S rRNA and several other 4S fragments are produced. Some of these 4S fragments may represent additional spacer tRNAs. One fragment, about 70 nucleotides long, arises from the 5′ end of the 17S precursor of 16S rRNA. Four or five other tRNAs are hydrogen-bonded to 30S rRNA as we prepare it; one or more of these tRNAs may also be a spacer tRNA. The enzymes that process tRNAs out of 30S rRNA are associated with ribosomes, but can be removed from them by washing in 0.2 M NH 4CI; the enzymes required for 5S rRNA processing remain bound to the 0.2 M NH 4CI-washed ribosomes. Treatment of 30S rRNA with purified RNAase III produces 6-8S fragments which contain the sequences of tRNA 2 Glu, tRNA 1B Ala and 5S rRNA.

Determination of 5'-triphosphate terminus of 16S ribosomal RNA precursor.

THE genes for the 23S and 16S rRNAs are physically linked1. Using the drugs rifampicin and actinomycin D, it has been shown that the 23S and 16S rRNA cistrons are co-transcribed and the promotor is proximal to 16S rRNA cistron2,3. Recent work with an Escherichia coli strain deficient in RNase III has provided further evidence for co-transcription, showing the presence of a long polycistronic RNA which could be cleaved to the immediate precursors of 23S and 16S rRNAs when treated with RNase III in vitro4. We have now studied the transcription of rRNA in E. coli using γ-32P-labelled triphosphates and have shown that the 16S rRNA precursor in our preparation has a 5′-pppA end and that in our system it is neither cleaved nor replaced by 5′-pA as observed by Takanami5. We have not found any detectable 5′-γ-32P-triphosphate end in the 23S rRNA species. This observation further verifies directly that both the 16S and 23S rRNAs belong to one transcriptional unit in the following order: promotor–16S–23S.

Short-lived, small RNAs in the cytoplasm of HeLa cells

Two methylated species of mammalian cytoplasmic RNA from HeLa cells are described. They migrate at about 74% (A) and 86% (B) of the speed of 5S rRNA on 10% polyacrylamide gel electrophoresis. Upon continuous incubation with 3H-uridine, when the rate of isotope incorporation into 5S rRNA ceases to be linear, the 3H levels of these species quickly decrease, indicating their short life. By “chase” with 5μg of actinomycin D/ml, their half lives are about 10 min. A and B appear to be coded by the nuclear genome, as ethidium bromide has little effect on their transcription. There must be a very brief nuclear processing step, if any, because A and B can be detected in the cytoplasm after 4 min of incubation with 3H-uridine, the shortest time tested. They do not seem to be part of the 45S precursor rRNA transcript, as their synthesis is not inhibited by 0.04 μg of actinomycin D/ml. Similar species have been found in hamster and mouse cells. Contamination by mycoplasmas could not be detected.

The behaviour of heterogeneous nuclear ribonucleic acid in partially and completely denaturing conditions.

It is shown that the heterogeneous nuclear RNA (HnRNA) synthesized in the presence of actinomycin and at low and high temperatures sediments in low-ionic-strength sucrose gradients between the rRNA components, similar to the unmethylated RNA synthesized under ;step-down' conditions. If the ionic strength is increased then the HnRNA sediments more rapidly than 28S rRNA, with a large proportion about the 45S precursor rRNA position. Initially this was thought to be due to aggregation of the HnRNA; however, centrifugation and electrophoresis in completely denaturing conditions suggest that the molecular weight of this species of RNA is very large The experiments reveal that HnRNA is conformationally unstable relative to the nucleolar RNA and that the slower sedimentation rate of HnRNA in 5mm-EDTA-Tris base-sucrose gradients reflects the greater expansion of the HnRNA relative to the nucleolar RNA. The implications of this finding are discussed.

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids.

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.

RNA and protein elongation rates in Saccharomyces cerevisiae.

The RNA elongation rate has been measured in yeast by the kinetics of appearance of radioactivity in the different molecular weight classes by the method first developed by Bremer and Yuan (1968). Despite the limitations caused by the breakdown of the 35s rRNA precursor, an estimate of 29 to 38 nucleotides/second at 30° has been obtained for the RNA elongation rate. The protein elongation rate has been calculated by the method of Maaloe and Kjeldgaard (1966) which consists of dividing the number of amino acids polymerized into protein per unit of time by the number of active ribosomes. This has given values of 7 to 9 amino acids/second at 30°.

A spectrophotometric study of the secondary structure of precursor ribosomal ribonucleic acid from Krebs ascites-tumour cells.

The spectrophotometric analysis of 45S precursor rRNA shows that it contains more G and C residues than does mature 28S or 18S rRNA. The helical content and the length of double-helical segments in 45S and 28S rRNA are similar.

The site of action of 5-fluoroorotic acid on the maturation of mouse liver ribonucleic acids

The action of 5-fluoroorotic acid on the in vivo [ 14C]orotate labelling of mouse liver RNA was studied. The incorporation of 5-fluoroorotic acid into polynucleotide chains has the following effects: 1. 1. The labelling of nuclear and cytoplasmic 28-S and 18-S rRNA is almost completely inhibited, while the transcription of 45-S precursor rRNA and its early processing to 32-S and 21-S rRNA is not affected by the drug. Therefore, 5-fluoroorotic acid blocks selectively the last steps of rRNA maturation. The synthesis of nuclear 5-S rRNA is only slightly inhibited, but its transport to cytoplasmic ribosomes is blocked. 2. 2. The labelling of heterogeneous nuclear RNA and messenger-like RNA of cytoplasmic polysomes and free postmicrosomal ribonucleoproteins is not affected by 5-fluoroorotic acid. The drug causes the appearance of rapidly labelled degradation products in the cytoplasmic soluble RNA fraction, which indicates enhanced degradation of messenger-like RNA. 3. 3. Administration of 5-fluoroorotic acid does not inhibit either the synthesis of nuclear precursor tRNA, its conversion into mature nuclear tRNA or the appearance of free and ribosome-bound tRNA in the cytoplasm. It is suggested that the interference of 5-fluoroorotic with a definite step of rRNA maturation acid may be useful in the elucidation of the molecular mechanisms of this process and of the biological effects of the drug.

Synthesis and processing of nuclear RNA in human monocytes after phagocytosis of polystyrene particles

Nuclear RNA was extracted from human monocytes separated by bovine serum albumin gradients. Sedimentation profiles yielded patterns of undegraded RNA. The turnover of nascent RNA in resting cells appeared to be slow. In monocytes engulfing latex particles an increase in the synthesis of 45-S rRNA precursor and an accelerated processing into mature 28-S and 18-S moieties occurred. Chase experiments indicated a higher rate of delivery of mature ribosomal RNA to the cytoplasm in these activated cells.

Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of Bacillus thuringiensis

The exotoxin of Bacillus thuringiensis, a structural analogue of ATP, is a strong inhibitor of in vivo RNA synthesis in mice liver. Administration of low doses of exotoxin causes the following changes in the labelling of liver RNA species. 1. 1. The synthesis of nuclear rRNA, including the 45-S precursor rRNA, is inhibited almost completely, while the labelling of nuclear heterogeneous “DNA-like” RNA is inhibited only to about 50 % of the level in control mice. 2. 2. The labelling of cytoplasmic 28-S and 18-S rRNA is inhibited to about 10 % of control levels. The same degree of inhibition is observed with rRNA extracted from isolated microsomes, ribosomes or postmicrosomal ribonucleoproteins. Cytoplasmic mRNA from microsomes and postmicrosomal ribonucleoproteins is inhibited to the same extent as nuclear and cytoplasmic rRNA. 3. 3. The labelling of 5-S rRNA extracted from ribosomes is decreased to about 10 % of control levels, identical to the inhibition of the other rRNA fractions studied. On the other hand, the synthesis of tRNA is relatively resistant to exotoxin, showing only about 50 % inhibition. The results are discussed and it is suggested that 5-S rRNA may be important in the nucleoplasmic control of rRNA synthesis and ribosome maturation.

Ribosomal RNA turnover in contact inhibited cells.

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson6 determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with 3H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of 3H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”7 of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations6. (2) 45S RNA labelled with a 20 min pulse of 3H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.

Defective control of ribosomal RNA processing in stimulated leukemic lymphocytes.

The kinetics of ribosomal RNA transcription and processing were assessed in chronic lymphocytic leukemia (CLL) lymphocytes during the initial phases of their delayed response to phytohemagglutinin. When compared to cultures of normal lymphocytes, CLL cultures developed normal augmentations in 45S rRNA precursor transcription and cleavage after a 1 hr incubation with PHA. However, failure to conserve 18S RNA subunits persisted in the CLL cultures. Subsequently the PHA-induced progressive rise in 45S RNA transcription became aborted and the over-all rate of RNA synthesis lagged far behind the levels attained by normal cultures incubated with PHA for 48 hr. CLL cultures responding to PHA in a delayed fashion exhibited efficient conservation of 18S RNA at 168 hr. In normal cultures, PHA-induced conservation of 18S RNA appeared to be independent of any effect on 45S ribosomal RNA precursor transcription. Therefore, the sluggish growth response of CLL lymphocytes was associated with a defect in one of the important mechanisms regulating assembly of new ribosomes.

Primary structural relationship of p16 to m16 ribosomal RNA.

IMMATURE ribosomes contain RNAs which can be distinguished from the molecules present in mature ribosomes1. The precursor of 16S rRNA (p16) can be identified in pulse labelled cells2–4; the conversion of p16 to mature 16S rRNA (m16) occurs during the latter stages of ribosome assembly and demands concomitant protein synthesis1. We have compared oligonucleotide fingerprints of E. coli p16 and m16 rRNA mixtures, labelled respectively with32P and3H or32P and33P and now report that the precursor is of 10% greater molecular weight than the mature molecules. Identifiable sequences are cleaved from the precursor during the maturation process.

Sequence studies on precursor 16S ribosomal RNA of Escherichia coli.

THE 16S and 23S rRNAs of E. coli are known to be synthesized through intermediates of marginally. higher S values (about 17S and 24S). Although various authors1–3 have reported the presence of these precursors, no sequence studies have been presented. Nevertheless, there is strong but indirect evidence3 that the precursor 16S rRNA (p16) is about 200 residues longer than 16S rRNA (ml6). In contrast, the evidence suggests that the precursor 23S rRNA has few or no further residues.

Cinétique de l'incorporation d'uridine ( 3H) dans des fractions subnucléolaires de cellules d'hépatome ascitique du rat

Summary The kinetics of (3H) uridine labelling of elements resulting from the lysis of the nucleolar apparatus after solubilization of chromatin by heparine is studied ; this enabled us to evidence an early-labelled fraction distinct from the classically described ones in which the maturation of the 45S rRNA precursor takes place. This fraction had not been observed yet. This fraction could correspond either to the material still bound to intra-nucleolar chromatin and removed by heparine, or to the fibrillar elements known to be labelled before granular RNP during the synthesis of rRNAs precursors.

Effect of serum on the synthesis of RNA and of DNA in the cultured lens epithelium of the chick embryo: Initiation of lens fiber formation in vitro

1. 1. The effect of fetal calf serum on RNA and DNA synthesis in the 6-day chick embryo lens epithelium was investigated by electrophoresis, sucrose gradient analysis and CsCl buoyant density centrifugation. The cells in the excised lens epithelium elongate in culture when the medium is supplemented with fetal calf serum. 2. 2. Fetal calf serum stimulated the incorporation of [2,8- 3H 2]adenosine into polydisperse and 4-S RNA's by about 2-fold and into 28-S and 18-S ribosomal RNA's (rRNA) by approx. 4-fold in 3 h. These values were normalized on the basis of the specific activity of the intracellular pool of [ 3H]ATP. Experiments with actinomycin D (dactinomycin) showed that the greater incorporation was not due to slower degradation of RNA. 3. 3. After 3 h in the presence of l-[ Me- 3H]methionine, fetal calf serum increased the labeling of rRNA by about 3.8-fold. Fetal calf serum stimulated the rate of methylation of 45-S precursor rRNA and the accumulation of methylated 32-S, 28-S and 18-S RNA's while affecting neither the extent nor the rate of labeling of the S-adenosylmethionine pool. 4. 4. Fetal calf serum did not stimulate the rate of DNA synthesis, but possibly decreased it. 5. 5. These results show that the initiation of lens fiber formation in vitro by fetal calf serum is associated with an increase in the synthesis of all types of RNA, especially of rRNA.