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PubMed1999

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule.

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.

Bacteriophage T7↗

PubMed1995

U14 base-pairs with 18S rRNA: a novel snoRNA interaction required for rRNA processing.

U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA.

Anti-Bacterial Agents↗

PubMed2005

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting.

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.

DNA Fingerprinting↗

PubMed1994

Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes.

We have previously shown that the 23S rRNA of Salmonella strains is highly fragmented by specific enzyme cleavages. In this article, we report that 23S rRNA of Salmonella strains is rapidly degraded as the cells enter the stationary phase. More than 90% of the 23S rRNA is degraded when the cells reach the stationary phase. The rate of degradation of 23S rRNA correlated with its degree of fragmentation. This degradation is probably mediated by newly synthesized protein factor(s), since treatment with chloramphenicol or rifampin inhibits the rRNA degradation. We propose that degradation of 23S rRNA is a novel mechanism in the regulation of the bacterial 23S rRNA and ribosome concentration and that this additional regulatory mechanism provides some selective advantage to cells.

Cell Division↗

PubMed1989

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.

Base Sequence↗

PubMed1990

18 S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells.

The effect of ionizing radiation (137Cs) on processing and transport of ribosomal RNA (rRNA) was studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. This approach permits kinetic analysis of processing of 45 S rRNA (radiolabeled predominantly prior to irradiation) into its 28 S and 18 S rRNA daughter species following irradiation. By this technique, we have recently demonstrated an increase in the normal 28 S:18 S rRNA stoichiometric ratio of 1:1 to as high as 1.6:1 during the interval 5 to 20 h following irradiation of HeLa cells at greater than or equal to 7.5 Gy. Alterations in 28 S:18 S ratio were evaluated in greater detail at early times following irradiation, up to 2 h. The 28 S:18 S ratio was found to be maximal at 1 h after radiation, at about 2:1, following 5 or 10 Gy. Using a method for rapid separation of nucleus from cytoplasm, transport of rRNA from nucleus to cytoplasm was also evaluated during this period. Despite an increase in the rate of 45 S rRNA processing, as well as an increased 28 S:18 S ratio, no alterations in transport from nucleus to cytoplasm were detected. This lack of transport alteration suggests that accumulation of excess 28 S rRNA is restricted to the nucleus, where it may represent an early step in the process of radiation-induced cell killing.

Biological Transport↗

PubMed1995

[Study on the characteristics of molecular epidemiology for Salmonella typhi isolated in China. II. Analysis of 16S rRNA gene restriction patterns (16S rRNA GRP) of S. typhi isolated in China].

16S rRNA gene, a highly conservative gene in molecular evolution, was labeled with digoxigenin-11-dUTP by PCR. Using it as a probe, we investigated the 16S rRNA gene restriction patterns (16S rRNA GRP) of S. typhi after their chromosomes were digested with Pst I. Results show that the Pst I 16S rRNA GRP appears obvious polymorphism in strains of distinctive sources. The fragments containing 16S rRNA gene are sized 7.0-26.5kb in 16S rRNA GRP of Pst I. 119 strains can be divided into 38 ribotypes. Most of the strains causing outbreaks from Dalian in 1990 and strains isolated during the pandemic from Yili, Xinjiang in 1991 have the same ribotype; some ribotypes contain epidemic strains which are from different geographic areas; some strains from sporadic cases have their unique ribotypes. We also find the 16S rRNA GRP with Pst I of S. typhimurium rather different from that of S. typhi. Data from further analysis of Pst I ribotypes by mean-linkage clustering method show that pandemic strains, outbreak strains and epidemic strains cluster at 0.55 in genetic distance; nonepidemic strains and strains from sporadic cases gather to form another cluster at 0.70 in genetic distance. Ribotype of strain 251, which was isolated forman asymptomatic carrier is different from the two clusters. The ribotype of S. typhimurium is apparently far distant, comparing with that of S. typhi.

China↗

PubMed2005

Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard.

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M. fortuitum infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.

Adaptation, Physiological↗

PubMed1996

Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon.

Gene-inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon. This mutant of Mycobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector-borne gene. By homologous recombination with the chromosome a plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of mutated ribosomes in the cell. As a first result we demonstrate that the single allelic knock-out strain allows for isolation of rRNA mutants with a drug-resistant phenotype, circumventing the problem of recessivity which prohibits the isolation of such mutants in organisms with multiple rRNA operons. Subsequently, by allelic exchange experiments, it was demonstrated that the rRNA mutation found indeed confers drug resistance in vivo. This system provides intriguing potential for the study of the structure and function of ribosomal RNAs.

Alleles↗

PubMed1998

Inhibition of ribosomal subunit association and protein synthesis by oligonucleotides corresponding to defined regions of 18S rRNA and 5S rRNA.

Strong complementarity between a conserved sequence near the 3' end of 18S (16S) rRNA of the small ribosomal subunit and a conserved sequence in the 5S rRNA of the large ribosomal subunit supported the suggestion that base-paired interaction between the two RNA molecules could be responsible for the reversible association of ribosomal subunits during protein synthesis. If this were true then oligonucleotides corresponding to defined regions of the 18S and 5S rRNAs should have profound effects on the association of ribosomal subunits and protein synthesis. In this report we show that oligonucleotides, corresponding to a defined region of eukaryotic 18S rRNA, when bound to wheat embryo 60S ribosomal subunits, inhibited association with 40S ribosomal subunits and also inhibited in vitro protein synthesis. Similarly oligonucleotides corresponding to a defined region of 5S rRNA when bound to 40S ribosomal subunits also inhibited the formation of 80S ribosomes and in vitro protein synthesis. The minimum sequences responsible for the inhibition of ribosomal subunit association and in vitro protein synthesis corresponded to the 5' strand of the m2(6)A m2(6)A hairpin structure near the 3' end of 18S rRNA and nucleotides 91-100 of 5S rRNA which are complementary to each other. Sequences at identical positions of Escherichia coli 16S and 5S rRNAs are also complementary to each other.

Base Composition↗

PubMed1994

A chloroplast group I intron undergoes the first step of reverse splicing into host cytoplasmic 5.8 S rRNA. Implications for intron-mediated RNA recombination, intron transposition and 5.8 S rRNA structure.

The chloroplast 23 S rRNA gene of Chlamydomonas reinhardtii contains a self-splicing group I intron, Cr.LSU. Incubation of total RNA from C. reinhardtii with [alpha-32P]GTP produces a GTP-labeled RNA that is derived from Cr.LSU, but is approximately 95 nucleotides longer (and referred to as IA). We show that IA is derived from an intermolecular reaction of Cr.LSU with cytoplasmic 5.8 S rRNA; the 3'-terminal G of the intron becomes ligated to A64. This reaction, which was reproduced using synthetic RNAs, is identical to the first step of reverse splicing. The second step of reverse splicing did not occur with any efficiency, even though both P1 and P10-like helices can form between the reactive region of 5.8 S rRNA and the IGS of Cr.LSU. Cloning of IA provided a chimeric Cr.LSU precursor with the normal 3' exon substituted by 95 nucleotides of 5.8 S; this precursor spliced poorly despite having a potential P10 helix (between the 3'-exon and the IGS). The inefficient reverse splicing of Cr.LSU into 5.8 S RNA and poor forward splicing of the chimera may be explained by competition between helices P1 (between the 5'-exon and IGS) and P10; competition between P1 and P10 is apparently not a factor in forward splicing of the wild-type LSU precursor. The sequence of the 5.8 S gene of C. reinhardtii was determined, and the published RNA sequence revised. A secondary structure model of C. reinhardtii 5.8 S rRNA was proposed incorporating the requirement for base-pairing to the IGS of Cr.LSU. The resulting structure resembles that recently proposed for yeast 5.8 S rRNA, and has considerable advantages over previous models of C. reinhardtii 5.8 S rRNA. These data describe a novel example of a naturally occurring ribozyme reacting with a naturally occurring RNA of the same cell; implications for ribozyme-mediated RNA recombination and intron transposition via reverse splicing are discussed.

Animals↗

PubMed1996

Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance.

Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247. This is close to a short open reading frame in the 23 S rRNA that encodes a pentapeptide (E-peptide) whose expression in vivo renders cells resistant to erythromycin. Therefore, a possible mechanism of resistance caused by domain II mutations may be related to an increased expression of the E-peptide. To test this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore, the erythromycin resistance determinant in the mutants was shown to be confined to a small 23 S rRNA segment containing the coding region and the ribosome binding site of the E-peptide open reading frame. It thus appears that the domain II mutations mediate erythromycin resistance by increasing expression of the 23 S rRNA-encoded E-peptide.

Base Sequence↗

PubMed1987

An intron in the 23S rRNA gene of the Chlorella chloroplasts: complete nucleotide sequence of the 23S rRNA gene.

A 243 bp intron was found within the 23S rRNA gene of the unicellular green alga Chlorella ellipsoidea. This intron is A+T-rich (63.7%) compared with the 23S rRNA (50.5%) and is located in domain II of the 23S rRNA. In contrast to rRNA introns so far known, this intron is considerably small and does not possess features of group I introns in spite of its possible folded secondary structure; this is a new type rRNA intron. The complete nucleotide sequence of the 23S rRNA gene (2,965 bp) was also compared with that of tobacco chloroplasts and E. coli.

Base Sequence↗

PubMed1981

Evidence on the conformation of HeLa-cell 5.8 S rRNA during interaction with 28 S rRNA.

The sites and extents of bisulphite-mediated deamination of cytidine residues in HeLa-cell 5.8 S rRNA within the 5.8 S / 28 S rRNA complex have been determined. Comparison of these data with equivalent data for isolated ('unbound') 5.8 S rRNA suggests that the conformation of several cytidine residues, including inter alia those within the 5'- and 3'-terminal sequences, differs in the two forms of 5.8 S rRNA. We therefore conclude that the sequences comprising stem 'a' of the isolated 5.8 S rRNA are involved in hydrogen-bonding to 28 S rRNA in the 60 S ribosomal subunit, but that the interactions are more subtle and complex than those proposed in earlier models.

Base Sequence↗

PubMed1996

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid: implication for diversification of insect rRNA.

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 bp in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those of Drosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect.

Animals↗

PubMed1991

Mycoplasma gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA.

Southern hybridization and cloning experiments revealed existence of 3 regions hybridizing with 16 S rRNA and 2 regions hybridizing with 23 S and 5 S rRNA in Mycoplasma Gallisepticum strain S6 genome thus forming 4 separate contiguous regions. One set of a putative rRNA genes is organized classically for eubacteria order 16 S-23 S-5 S. The other two 16 S rRNA and the other one 23 S-5 S rRNA hybridizing regions are separated from each other and from the complete rRNA operon for a distance of more than 6 kb.

Base Sequence↗

PubMed1987

Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation.

It was shown that induction of rRNA (and ribosome) synthesis from the lambda PL promoter/operator by temperature shift-up causes a repression of rRNA and tRNA synthesis from chromosomal genes. We have carried out experiments using a similar conditional rRNA gene expression system in which a mutational alteration was introduced in the anti-Shine-Dalgarno region at the 3'-end of the 16 S rRNA gene. It was found that the repression observed with the wild-type gene was largely abolished by the mutation. It appears that ribosomes inefficient in translational initiation are unable to cause feedback regulation of rRNA synthesis. It is suggested that the cell regulates rRNA (and tRNA) synthesis by monitoring the production of ribosomes, and that this monitoring is apparently carried out through their activity in the initiation (and perhaps subsequent steps) of translation.

Escherichia coli↗

PubMed1993

Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly.

We have generated temperature-sensitive lethal point mutations in the small nucleolar RNA-associated protein fibrillarin (encoded by the NOP1 gene in yeast) and analyzed their effects on ribosome synthesis. The five alleles tested all prevent synthesis of normal ribosomes, but in dramatically different ways. At the non-permissive temperature, the nop1.2 and nop1.5 alleles prevent synthesis of both 18S and 25S rRNA and all pre-rRNA species except the 35S primary transcript. In contrast, the nop1.3, nop1.4, and nop1.7 alleles do not strongly impair processing. In nop1.3 strains, nucleolar methylation of pre-rRNA is strongly inhibited; late, cytoplasmic methylation of 18S rRNA and tRNA methylation continue. The nop1.4 and nop1.7 alleles result in the synthesis of cytoplasmic 60S ribosomal subunits with strongly aberrant mobilities on sucrose gradients even at the permissive temperature, owing to the impairment of a late step in ribosome assembly. Thus, all major posttranscriptional activities in ribosome synthesis, pre-rRNA processing, pre-rRNA modification, and ribosome assembly are dependent on fibrillarin.

Base Sequence↗