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At least 19 recordsLinked to original sources
PubMed1991

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.

Animals↗
PubMed1983

Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.

We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

Animals↗
PubMed1985

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA.

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).

Animals↗
PubMed1987

An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon.

We succeeded in identifying a promoter element within 200 base pairs upstream a transcriptional unit comprising only a 23S rRNA, 5S rRNA and a tRNA(gly) gene in Thermus thermophilus HB8 [1, 2]. This element shows a high degree of homology to the -35 and -10 consensus sequences for promoters described for Escherichia coli [3, 4]. The promoter activity was measured by the induction of the synthesis of functional chloramphenicol acetyltransferase in Escherichia coli. A region located at the transcriptional start, rich in guanosines and cytidines, is very similar in sequence to the one believed to be under stringent control in stable RNA and ribosomal protein genes of Escherichia coli [5]. Employing nuclease S1 protection we were able to determine the in vivo start of transcription, which was identical with the in vitro start using Escherichia coli RNA-polymerase. Furthermore we identified sequences in the region following the origin of transcription, which are homologous to sections in Escherichia coli rrn promoter-leader regions responsible for antitermination. Our finding of a promoter immediately preceding a 23S/5S rRNA operon proves a transcriptional decoupling of the 16S rRNA genes, a situation so far unprecedented among prokaryotes.

Acetyltransferases↗
PubMed1983

Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons.

We have tested a model for global ribosome biosynthesis by examining the effects of increased gene dosage on the synthesis of rRNA. Increasing gene dosage does not lead to a significant increase in total rRNA transcription; i.e., rRNA synthesis from individual rRNA operons is reduced to keep total rRNA production unchanged. In contrast, when the plasmid-encoded rRNA operons used to increase gene dosage contain deletions within the rRNA coding region, rRNA transcription is gene-dosage-dependent; i.e., rRNA regulation is relieved. We find that the syntheses of most, if not all, tRNAs are also subject to the same controls as rRNA transcription. We conclude that the production of functional rRNA is monitored by the regulatory system that controls rRNA and tRNA transcription. We propose that rRNA and tRNA are negatively controlled by products of rRNA operons and discuss evidence suggesting that ribosomes are the key element involved in the postulated feedback regulation.

Chromosome Deletion↗
PubMed1982

The nucleotide sequence at the 3'-end of Neurospora crassa 18S-rRNA and studies on the interaction with 5S-rRNA.

The sequence of more than 100 nucleotides at the 3'-end of Neurospora crassa 18S-rRNA was determined by chemical sequencing techniques. Extensive homologies with 18S-rRNA from other eukaryotes were found. Inspection of the nucleotide sequence at the 3'-end of N. crassa 5S-rRNA revealed the presence of sequences complementary to a region near the 3'-terminus of 18S-rRNA. Under the appropriate conditions a complex was formed between 18S-rRNA and 5S-rRNA (Tm 53 degrees C). Interaction was detected between 5S-rRNA and a specific 3'-terminal fragment from 18S-rRNA and between 18S-rRNA and a specific 3'-terminal fragment from 5S-rRNA. These findings are consistent with the idea that intermolecular base-pairing between nucleotides at the 3'-ends of 18S-rRNA and 5S-rRNA may be functionally important within the ribosome. Further investigation revealed that this intermolecular base-pairing is not essential for ribosome stability.

Animals↗
PubMed1988

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. II. Unique properties of an operon containing a minor 5 S rRNA gene.

This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes. The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J. (1988) J. Biol. Chem. 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B. subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA. Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B. subtilis 168 genome. S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter. No promoters have been found immediately 5' to any of the major 5 S rRNA species in B. subtilis rRNA operons. S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism. S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon. A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation. A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs. Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter. However, the last tRNA gene is primarily under the control of its own unique promoter.

Bacillus subtilis↗
PubMed1991

Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome.

The locations of close encounter between ribosomal RNA (rRNA) and messenger RNA (mRNA) were determined by photochemical cross-linking experiments that employ an artificial mRNA, 51 nucleotides long, containing 14 U residues that were randomly substituted by 1-4 4-thiouridine (s4U) residues. The mRNA was bound to 70S ribosomes or 30S subunits and then was irradiated at 366 nm to activate cross-linking between the s4U residues and rRNA. Cross-linking occurred to both 16S rRNA and 23S RNA. The rRNA was then analyzed by a series of reverse transcriptase experiments to determine the locations of cross-linking. Twelve sites in the 16S rRNA and two sites in the 23S rRNA have been detected. In the 16S rRNA, two of the sites (U1381, C1395) are in the middle part of the secondary structure close to position C1400, and the remaining sites (G413, U421, G424; A532; G693; U723; A845; G1131/C1132; G1300; G1338) are distributed between six regions that are peripheral in the secondary structure. In the 23S rRNA, one site (U1065) is located in the GTPase center close to A1067, the site of thiostrepton-resistance methylation in domain II, and the other site (U887) is located a short distance away also in domain II. The distribution of these rRNA sites in the ribosome specifies an mRNA track that is consistent with other information. In addition, some of the contact points represent new constraints for the three-dimensional folding of the rRNA.

Base Sequence↗
PubMed1987

Clones of human ribosomal DNA containing the complete 18 S-rRNA and 28 S-rRNA genes. Characterization, a detailed map of the human ribosomal transcription unit and diversity among clones.

We have isolated several new clones of human ribosomal DNA. Each clone contains part of the external transcribed spacer, a complete 18 S-rRNA gene, the internal transcribed spacers, a complete 28 S-rRNA gene and a short downstream flanking region. We present a detailed map of the human ribosomal transcription unit with the locations of numerous useful restriction sites. In particular, a unique NheI site in the 5.8 S-rRNA gene enabled this gene to be mapped with respect to the 18 S-rRNA and 28 S-rRNA genes. The human 45 S-rRNA coding region is approx. 13,000 nucleotide residues long, of which the external transcribed spacer comprises approx. 3700 nucleotide residues and the first and second internal transcribed spacers comprise approx. 1070 and 1200 nucleotide residues respectively. A partial survey for sites of variation between clones has revealed a single point of variation among 18 S-rRNA gene sequences (a T/C variation at position 140), several sites of length variation in the regions of the transcribed spacers closely flanking the 18 S-rRNA genes, and some sites of length variation among 28 S-rRNA genes. Most of these sites of variation are associated with simple sequence tracts and are in regions that are known to undergo relatively rapid evolutionary divergence. In particular, the sites of variation among 28 S-rRNA genes occur in G + C-rich tracts whose lengths vary among vertebrates and that can be correlated with extensive hairpin structures previously observed by electron microscopy. Each of the clones so far surveyed in detail differs from the others in one or more respects.

Base Sequence↗
PubMed1985

Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage lambda. By analyzing pulse-labeled RNA with an RNA.DNA filter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoter-proximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage lambda system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears that E. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons.

Bacteriophage lambda↗
PubMed1975

Wheat-embryo ribonucleates. IV. Factors that influence the formation and stability of a complex between 5S rRNA and 18S rRNA.

Under the conditions used in this study, wheat-embryo 5S rRNA complexes with its homologous 18S rRNA from wheat embryos and with heterologous 18S rRNA from other eukaryotic source materials such as yeast, L cells, and HeLa cells, but it does not complex with heterologous 16S rRNA from a prokaryote such as Escherichia coli or with homologous or heterologous 26S (23S) rRNA of either eukaryotic or prokaryotic origin. If a solution of wheat-embryo rRNA is simply made 0.3 M with respect to NaCl and then heated at 60 degress C for 3 min before quick cooling to room temperature (ca. 20 degrees C), there is both preferential and efficient complex formation between 5S and 18S rRNA. The 'laboratory-prepared' complex between wheat-embryo 5S rRNA and its homologous 18S rRNA is more thermostable in 0.1 M NaCl solution than is the 'natural' complexes 'melt' over a narrow range of temperature. The possible physicochemical and physiological importance of both homologous and heterologous rRNA complexes is the subject of a brief discussion.

Binding Sites↗
PubMed1991

Evidence for a Competitive-Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA.

We have previously shown that a 5'-terminal region of mouse 5 S rRNA can base-pair in vitro with two distinct regions of 18 S rRNA. Further analysis reveals that these 5 S rRNA-complementary sequences in 18 S rRNA also exhibit complementarity to the Kozak consensus sequence surrounding the mRNA translational start site. To test the possibility that these 2 regions in 18 S rRNA may be involved in mRNA binding and translational initiation, we have tested, using an in vitro translation system, the effects of DNA oligonucleotides complementary to these 18 S rRNA sequences on protein synthesis. Results show that an oligonucleotide complementary to one 18 S rRNA region does inhibit translation at the step of initiation. We propose a Competitive-Displacement Model for the initiation of translation involving the intermolecular base-pairing of 5 S rRNA, 18 S rRNA and mRNA.

Animals↗
PubMed1979

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, der Boer and Nomura, 1979). The first two start sites are 283 and 291 bp preceding the mature 16S rRNA (m16S rNA) 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 or rrnD and rrnX, determined by Young and Steitz (1979), and some common features are discussed.

Base Sequence↗
PubMed1979

Identification of initiation sites for the in vitro transcription of rRNA operons rrnE and rrnA in E. coli.

The transcription initiation sites of E. coli rRNA operons were determined using various DNA fragments derived from transducing phage lambda metA20 carrying rrnE and from hybrid plasmid pLC19-3 carrying rrnA. In vitro transcription products were analyzed for their 5' end sequences and their oligonucleotide compositions. The results are in full agreement with the nuceotide sequences of the DNA templates described in an accompanying paper (de Boer, Gilbert and Nomura, 1979) and allow us to make the following conclusions. First, there are two transcription, start sites on each of the rRNA operons; they are 109 bp apart in the case of rrnE and 117 +/- 1 bp aprart in rrnA. Second, the first start site is 283 bp upstream from the m16S rRNA coding region in the case of rrnE, while is 291 bp upstream in rrnA. Initiation starts with ATP in both cases. Finally, the second start sites are 174 and 174 +/- 1 bp from the m16S rRNA genes in rrnE and rrnA, respectively. Initiation starts with CTP in both cases. We have also shown that in the present in vitro transcription system, guanosine tetraphosphate (ppGpp) inhibits the synthesis of full-sized RNAs from both start sites in each rRNA operon.

Base Sequence↗
PubMed1982

Sequence and secondary structure of mouse 28S rRNA 5'terminal domain. Organisation of the 5.8S-28S rRNA complex.

We present the sequence of the 5' terminal 585 nucleotides of mouse 28S rRNA as inferred from the DNA sequence of a cloned gene fragment. The comparison of mouse 28S rRNA sequence with its yeast homolog, the only known complete sequence of eukaryotic nucleus-encoded large rRNA (see ref. 1, 2) reveals the strong conservation of two large stretches which are interspersed with completely divergent sequences. These two blocks of homology span the two segments which have been recently proposed to participate directly in the 5.8S-large rRNA complex in yeast (see ref. 1) through base-pairing with both termini of 5.8S rRNA. The validity of the proposed structural model for 5.8S-28S rRNA complex in eukaryotes is strongly supported by comparative analysis of mouse and yeast sequences: despite a number of mutations in 28S and 5.8S rRNA sequences in interacting regions, the secondary structure that can be proposed for mouse complex is perfectly identical with yeast's, with all the 41 base-pairings between the two molecules maintained through 11 pairs of compensatory base changes. The other regions of the mouse 28S rRNA 5'terminal domain, which have extensively diverged in primary sequence, can nevertheless be folded in a secondary structure pattern highly reminiscent of their yeast' homolog. A minor revision is proposed for mouse 5.8S rRNA sequence.

Animals↗
PubMed1981

The 3'-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3'-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.

Animals↗
PubMed1984

Level of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA operons in Escherichia coli.

We have recently proposed a model for the negative feedback control of rRNA and tRNA synthesis in Escherichia coli by products of rRNA operons or their derivatives (e.g., nontranslating ribosomes) (S. Jinks-Robertson, R.L. Gourse, and M. Nomura, Cell 33:865-876, 1983). In this paper, we examined the following questions. (i) Are the spacer tRNAs carried within rRNA operons the products responsible for the regulation of rRNA and tRNA transcription? (ii) Are tRNAs capable of regulating their own syntheses? We measured tRNA accumulations in cells containing plasmids with intact or defective rRNA operons or with tRNA operons. From the results obtained, we conclude that neither the tRNAs encoded within rRNA operons nor the tRNAs encoded in non-rRNA operons are capable of controlling rRNA or tRNA transcription. Therefore, the products responsible for the initial step leading to rRNA and tRNA regulation are rRNAs (or their derivatives).

Aspartate-tRNA Ligase↗
PubMed1987

The secondary structure of human 28S rRNA: the structure and evolution of a mosaic rRNA gene.

We have determined the secondary structure of the human 28S rRNA molecule based on comparative analysis of available eukaryotic cytoplasmic and prokaryotic large-rRNA gene sequences. Examination of large-rRNA sequences of both distantly and closely related species has enabled us to derive a structure that accounts both for highly conserved sequence tracts and for previously unanalyzed variable-sequence tracts that account for the evolutionary differences in size among the large rRNAs. Human 28S rRNA is composed of two different types of sequence tracts: conserved and variable. They differ in composition, degree of conservation, and evolution. The conserved regions demonstrate a striking constancy of size and sequence. We have confirmed that the conserved regions of large-rRNA molecules are capable of forming structures that are superimposable on one another. The variable regions contain the sequences responsible for the 83% increase in size of the human large-rRNA molecule over that of Escherichia coli. Their locations in the gene are maintained during evolution. They are G + C rich and largely nonhomologous, contain simple repetitive sequences, appear to evolve by frequent recombinational events, and are capable of forming large, stable hairpins. The secondary-structure model presented here is in close agreement with existing prokaryotic 23S rRNA secondary-structure models. The introduction of this model helps resolve differences between previously proposed prokaryotic and eukaryotic large-rRNA secondary-structure models.

Animals↗