Yeast rRNA Research Articles

Heat shock causes transient inhibition of yeast rRNA gene transcription

The heat-shock response in the yeast Saccharomyces cerevisiae includes transiently decreased production of the full-size pre-rRNA transcript. Here we have used quantitative hybridization of pulse-labeled RNA to cloned, immobilized sequences derived from the external transcribed spacer of yeast rDNA, coupled with determinations of relative changes in ATP pool specific activities, to show that the heat shock associated with the transfer of growing cells from 23 to 36 degrees C caused decreased transcription of the rRNA genes. This decrease in hybridization to DNA sequences complementary to the immediate 5' end of the pre-rRNA transcript suggests that the decreased transcription reflects decreased initiation of pre-rRNA transcription.

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10- to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10- to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.

Neurospora crassa nuclear genome contains analogy of Saccharomyces cerevisiae genes for ribosomal RNA processing.

Neurospora crassa wild type genome shows DNA sequences which are homologous to the sequences present in the rRNA processing genes of the yeast Saccharomyces cerevisiae. Five such processing genes from yeast, viz., RNA1 through RNA5, cloned in plasmid pBR322 were transformed in Escherichia coli strain LE392. Southern blots containing DNAs from these clones were restricted with several restriction endonucleases along with DNAs from lambda phage, rice (plant) and neuroblastoma (animal), were hybridized with 32P-labelled nick-translated N. crassa nuclear DNA under very stringent conditions. Autoradiograms of these blots revealed that four yeast rRNA processing genes (RNA1, RNA2, RNA3, and RNA4) showed homology with N. crassa nuclear DNA but such analogs were not found in DNAs representing prokaryotes, phages, higher plants and animals.

Organization of the 5.8S, 16?18S, and 23?28S ribosomal RNA genes of Cephalosporium acremonium

A 9.2 kb Pst1 restriction fragment, repeated tandemly head-to-tail in the genome, contains the 5.8S, 16–18S, and 23–28S ribosomal RNA (rRNA) genes of Cephalosporium acremonium, a filamentous fungus used at the industrial scale for production of cephalosporin antibiotics. These rRNA genes were located in Pst1 digests of C. acremonium genomic DNA using a hybridization probe that contained the 5.8S, 18S, and 25S rRNA genes from the yeast Saccharomyces cerevisiae. This probe was also used in screening a recombinant lambda library to identify phage carrying rRNA genes of C. acremonium. Yeast rRNA genes contained separately on individual 32P-labeled plasmids were used to demonstrate that a cloned 7.2 kb C. acremonium sequence, represented in the repeated 9.2 kb Pst1 fragment, contained DNA from the C. acremonium 5.8S, 16–18S, and 23–28S rRNA genes. These genes were ordered with the 5.8S gene located between the 16–18S and 23–28S rRNA genes. The order of the 16–18S, 5.8S, and 23–28S rRNA genes observed in C. acremonium is the same as that observed in rRNA repeats of many other lower eucaryotes, e.g. S. cerevisiae, Aspergillus nidulans, and Neurospora crassa.

Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.

A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes. The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx. 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207. Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA. Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping. These transcripts were initiated at the site used by the host rRNA genes, were approx. 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing.

Effect of seminal plasmin on rRNA synthesis in Saccharomyces cerevisiae

Seminal plasmin, the highly basic, antimicrobial protein, isolated from bull semen, was found to inhibit the transcription of ribosomal RNA in yeast. Protein synthesis and processing of rRNA remained unaffected. Seminal plasmin appears to be useful for studies of the biosynthesis of yeast rRNA in pulsechase experiments.

Transcription of an artificial ribosomal RNA gene in yeast.

We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain. The artificial gene comprised a fragment containing the sequence from position -207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre-rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position -36 to +101 relative to the 3' end of the 26S rRNA gene. The resulting construct was cloned into the yeast-Escherichia coli shuttle vector pJDB207. Both Northern blot hybridization and R-loop analysis of RNA from transformed Saccharomyces cerevisiae cells revealed a discrete transcript of the expected length. S1 nuclease mapping as well as primer extension analysis showed that the major proportion of the transcripts was initiated at exactly the same site as 37S pre-rRNA. These results show that the respective rDNA fragments contain the information for correct initiation of transcription and formation of the 3' end. A minor proportion of the transcripts was initiated at a number of sites between positions -1 and -100 upstream of the predominant start. The proportion and the pattern of these upstream starts is affected by the vector context of the artificial rRNA gene.

Cross-linking of the anticodon of Escherichia coli and Bacillus subtilis acetylvalyl-tRNA to the ribosomal P site. Characterization of a unique site in both E. coli 16S and yeast 18S ribosomal RNA.

The nucleotide residues involved in the cross-link between P site bound acetylvalyl-tRNA (AcVal-tRNA) and 16-18S rRNA have been identified. This cross-link was formed by irradiation of Escherichia coli or Bacillus subtilis AcVal-tRNA bound to the P site of E. coli ribosomes or by irradiation of E. coli AcVal-tRNA bound to the P site of yeast ribosomes. The three cross-linked RNA heterodimers were obtained in 10-35% purity by disruption of the irradiated ribosome-tRNA complex with sodium dodecyl sulfate followed by sucrose gradient centrifugation. After total digestion with RNase T1, and labeling at either the 5'- or the 3'-end, the cross-linked oligomers could be identified and isolated before and after photolytic splitting of the cross-link. One of the oligomers was shown to be UACACACCG, a unique rRNA nonamer present in an evolutionarily conserved region. This oligomer was found in all three heterodimers. The other oligomer of the dimer had the sequence expected for the RNase T1 product encompassing the anticodon of the tRNA used. The precise site of cross-linking was determined by two novel methods. Bisulfite modification of the oligonucleotide dimer converted all C residues to U, except for any cross-linked C which would be resistant by being part of a cyclobutane dimer. Sequencing gel analysis of the UACACACCG oligomer showed that the C residue protected was the 3'-penultimate C residue, C1400 in E. coli rRNA or C1626 in yeast rRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA.

The antiviral activity of ribosomal polynucleotides against encephalomyocarditis virus infection of mice.

Intraperitoneal administration of ribosomal RNA (rRNA) was found to protect mice against subsequent lethal infection by encephalomyocarditis (EMC) virus without induction of detectable amounts of circulating interferon. The nature of this effect was examined in terms of the types of natural polyribonucleotides which could afford such protection. rRNA prepared from E. coli was slightly more effective than chicken liver rRNA which was, in turn, more effective than yeast rRNA. 5S ribosomal RNA was not effective, whereas the slightly smaller 4S transfer RNA was as good as E. coli rRNA, suggesting that molecular size is not the sole criterion for the protective effect. The separated 16S and 23S E. coli rRNAs where each as effective as the unfractionated RNA. Anti-viral activity was lost after complete hydrolysis with alkali and nucleoside monophosphates were also inactive. Digestion of rRNA with pancreatic ribonuclease greatly decreased its antiviral activity whereas digestion with T1 ribonuclease had no effect indicating that fairly short oligonucleotides, but not of random nucleotide sequence, are active components in the protection of mice against infection by EMC virus. In vitro, no antiviral effect against EMC virus infection was observed in treatment of L cells under various conditions.

The stringent and relaxed phenomena in Saccharomyces cerevisiae.

We present here a detailed analysis of the effect of amino acid starvation and the addition of cycloheximide on RNA metabolism of yeast cells and spheroplasts. These effects have been studied at the level of uridine phosphorylation, methylation of rRNA, and biosynthesis of 35 S, 4 S, and 5 S RNA species. Amino acid starvation inhibits the phosphorylation of uridine assigned for RNA synthesis more than that for other metabolic processes. This implies that a salvage pathway for the synthesis of UMP and CMP is regulated by the rate of transcription and perhaps is localized in the nucleus. The rate of rRNA methylation is not coupled with the rate of transcription; therefore, quantitation of 35 S RNA synthesis (yeast rRNA primary transcript by [methyl-3H]methionine labeling is unreliable. Biosynthesis of 35 S RNA ceases immediately after the cells are transferred to an amino-acid-deficient medium; at a later time 4 S and 5 S RNAs are also inhibited. Therefore, coordination and noncoordination in the stringent response of these RNA species depend upon the time of starvation. Although addition of a small dose (less than 1.0 microgram/ml) of cycloheximide to starved yeast spheroplasts does not alter the rate of uridine phosphorylation, it increases the rate of entrance of uridine into total RNA. This effect is of greater magnitude in 4 S and 5 S than in 35 S RNA. Since the drug does not alter the rate of decay of 35 S RNA that takes place in starvation, it has a selective effect on transcription. A similar small dose, however, produces inhibition of transcription of all these RNA species in nonstarved conditions. This opposite effect of the drug appears to be a characteristic feature of RNA metabolism in eukaryotes.

Divergent transcription in the yeast ribosomal RNA coding region as shown by hybridization to separated strands and sequence analysis of cloned DNA

λ-yeast DNA hybrids containing the coding sequences for all four ribosomal RNAs from the yeast Saccharomyces cerevisiae were strand separated by the poly(rU, rG)-CsCl method. Hybridization of yeast rRNA to the separated strands indicated that the arrangement of the RNAs in the precursor transcript is 5′-18 S.5.8 S.25 S.3′ and that the 5 S rRNA, which hybridizes to the opposite strand, must be synthesized separately and in the opposite direction. The location and direction of the 5 S rRNA transcript was independently established by DNA sequence analysis. The presumptive promoter region adjacent to the 5′ end of the 5 S rRNA coding region was also sequenced.

Detection of yeast ribosomal RNA sequences in E. coli infected with hybrid bacteriophage

Yeast ribosomal DNA was inserted into Escherichia coli on a bacteriophage vector and the host cell RNA was then extracted and analyzed for the presence of yeast ribosomal RNA sequences. RNA complementary to yeast rDNA was detected by hybridization. The transcription of yeast rDNA was found to be independent of phage RNA synthesis and to occur on the same DNA strand as rRNA transcription in yeast. However, hybridization to restriction fragments of yeast rDNA suggested that the RNA species detected in E. coli differ somewhat from authentic yeast rRNA.

Viscometric behavior of yeast ribosomal RNA in mixed water-organic solvent solutions and its application to a simple determination of the molecular weight

Different samples of yeast rRNA have been studied as regards their molecular conformation in mixed water/organic solvents. In water/formamide, rRNA is in its native state at 20°C but undergoes a sharp melting transition around 43°C. On the contrary, in water/dimethylformamide and water/dimethylsulfoxide, there is already an important degree of denaturation at 20°C, which increases with organic solvent concentration. It is concluded that formamide disrupts the tertiary structure only; whereas the other solvents partially destroy the secondary structure of RNA. In water/formamide, the reduced viscosity displays a linear dependence upon concentration. In the presence of NaCl, there is only a decrease of intrinsic viscosity. With the other mixed solvents, the reduced viscosity curves present a sharp maximum, which progressively disappears when ionic strength is increased. These observations are interpreted on the basis of two effects: (i) a polyelectrolytic effect, bringing about an expansion of the semi-flexible chain in the absence of salt. (ii) An electroviscous effect producing an increase in intrinsic viscosity and a high positive dependence of reduced viscosity upon concentration. A correlation has been established between viscosity and sedimentation coefficient of various rRNA samples in 0.2 M NaCl. A relationship between sedimentation coefficient and molecular weight is also given in the same solvent. Advantage has been taken of the higher degree of extension of rRNA in some mixed water-organic solvents in order to establish a relationship between intrinsic viscosity ([η]) and molecular weight ( M r) of the form [ η] = kM a r. The empirical constants k and [α] have been determined for water 20% formamide and for 1 mM NaCl 20% dimethylsulfoxide . From the value of α, conclusions are drawn regarding the molecular conformation of rRNA in these solvents.

Modified sequences in yeast ribosomal RNA

26S and 17S yeast ribosomal RNA were digested with T 1 plus pancreatic ribonuclease and the products were fractionated by two-dimensional electrophoresis. Besides the expected standard products (type (Ap) n Np, where N is C, U or G) several non-standard products were found to be present in the digests. The latter products include methylated oligonucleotides and pseudouridylic acid (ψp)-containing fragments. The primary structure and molar frequency of these modified products were determined. They appeared to be present in approximately integral molar amounts. Several of these oligonucleotides contain more than one modification. The total number of ψp-residues in 26S and 17S yeast rRNA was estimated to be about 32 and 14, respectively.

Proflavine binding to yeast rRNA and ribosomes as related to structure.

AbstractProflavine binding experiments were carried out with yeast rRNA, native and “unfolded” ribosomes; the binding constants and the number of binding sites were calculated by a spectroscopic method. The study of the intercalation complexes by fluorescence and electric dichroism shows the intercalation binding sites to involve two subtypes of sites, which could be related to different nucleotide composition and secondary structure of the rRNA regions, i.e., binding sites located in the (A + U)‐rich single strands and binding sites located in the (G + C)‐rich double‐helical strands (fluorescence quenching sites). Electric dichroism of complexed proflavine is interpreted in terms of rRNA conformation within the ribosomes. The conclusions are in agreement with the ribosomal model of Cox and Bonanou and show that, according to this model, the base planes of the nucleotides are not all parallel in the native ribsome, but rather radiate around the folding axis of the ribonucleoprotein sheet.

On the mechanism of the biosynthesis of ribosomal RNA in yeast

The biosynthesis of 26-S and 17-S ribosomal RNA (rRNA) in the yeast Saccharomyces carlsbergensis was investigated. Four high-mol. wt. ribosomal RNA precursors with a size corresponding to sedimentation values of 42 S, 37 S, 32 S and 29 S were found to be present in this organism. These ribosomal precursor RNA's were purified and isolated by means of polyacrylamide gel electrophoresis of the nuclear RNA fraction. Structural comparison between the four purified ribosomal RNA precursors, pulse-labeled in vivo with various radioactive markers and the two rRNA components was made by competitive hybridization with homologous DNA. These studies showed that the ribosomal RNA precursors contain, besides sequences of mature rRNA, also nonribosomal stretches which are not conserved during the processing of 42-S RNA into rRNA but are removed in a number of steps. On the basis of these findings and the determined molecular weights of the four ribosomal RNA precursors and the two rRNA's, a scheme for the biosynthesis of rRNA in yeast is proposed.

Characteristics of the methylation in vivo of ribosomal RNA in yeast

The characteristics of the methylation in vivo of ribosomal RNA (rRNA) in yeast has been studied by examining the pattern of incorporation of labeled methyl groups into rRNA and into ribosomal precursor RNA. 1.1. Steady-state labeling with [Me-14C]methionine showed that the degree of methylation for 17-S and 26-S rRNA is very similar, being about 1 methyl group per 70 nucleotides for both rRNA's. The methylation of yeast rRNA was found to be mainly ribose methylation, amounting to 74 and 83% of the total methylation for 17-S and 26-S rRNA, respectively. Chromatographic analysis of the alkaline hydrolysates of the two separate rRNA's revealed that the distribution of the methyl groups along the polynucleotide chains is distinctly different for the two rRNA components.2.2. The kinetics of methylation was studied by simultaneous pulse-labeling with [Me-3H]methionine and [14C]uracil. The results showed that methylation starts already at the level of the first ribosomal RNA precursor, immediately after or at the time of its transcription. However, from the ratio of the incorporation of [3H]methyl and [14C]uracil, it could be inferred that additional methylation takes place at later stages of the maturation process of rRNA, presumably after the conversion of ribosomal precursor RNA to rRNA.3.3. By analysis of the distribution of the methyl label in rRNA after increasing periods of labeling with [Me-3H]methionine, it could be demonstrated that the additional methylation is base methylation, occurring in both 26-S and 17-S rRNA.

Studies on ribosomal RNA structure: II. Secondary structures in solution of rRNA and crystallizable fragments

1. 1. Parallel studies on the secondary structures in solution of rRNA and crystallizable fragments of rRNA by means of ultraviolet and infrared absorption spectroscopy are described. Fragmented and intact rRNA, which are obtained from yeast ribosomes, exhibit qualitatively similar melting properties in the ultraviolet, either in the presence or absence of formaldehyde, a denaturant of base pairing. Deuterium oxide solutions of both types of RNA also give rise to closely similar infrared absorption spectra in the 1550–1750 cm −1 region. 2. 2. The spectroscopic results show that fragmented and intact rRNA have secondary structures containing comparable and extensive amounts of hydrogen-bonded base pairs. The infrared results show further that at 30°, assuming only Watson-Crick pairing, the fragments have 66±6% bases paired ( 32±3% AU pairs, 34±3% GC pairs) while intact rRNA has 64±6% bases paired ( 29±3% AU, 35±3% GC). At 5°, the proportions of paired bases are increased to 76±7% and 69±7%, respectively, for fragments and intact rRNA. The thermal rupture of inter-base hydrogen bonds is essentially complete at 80°. 3. 3. The existence of ordered double helical regions in aqueous RNA fragments is consistent with the results of previous X-ray diffraction studies on crystalline fibers of this material which gave evidence for such structure in the solid state. Base pairing is therefore considered an integral part of the secondary structure of these fragments, rather than something arising from the process of crystallization. The extensive base pairing in yeast rRNA is in agreement with estimates of the double helicity of tobacco mosaic virus RNA, reticulocyte rRNA, 5-S RNA and tRNA.