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At least 19 recordsLinked to original sources

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↗

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↗

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↗

PubMed1999

Distinct types of rRNA operons exist in the genome of the actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon.

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete Thermomonospora chromogena. The genome of T. chromogena contains six rRNA operons (rrn), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon rrnB exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of rrnB is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between rrnB operon of T. chromogena and rrnA operon of another thermophilic actinomycete Thermobispora bispora. We propose that T. chromogena acquired rrnB operon from T. bispora or a related organism via horizontal gene transfer.

Actinomycetales↗

PubMed1999

An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria.

Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

Base Sequence↗

PubMed2006

A cyanobacterial strain with all chromosomal rRNA operons inactivated: a single nucleotide mutation of 23S rRNA confers temperature-sensitive phenotypes.

The presence of a multicopy chromosome, with each copy containing two rRNA operons (rrnA and rrnB), has been an obstacle to analysing mutated rRNA in Synechococcus PCC 7942. To create a system for expressing homogeneous mutated rRNA, the chromosomal rrn operons were sequentially inactivated and a final strain was successfully obtained with all the chromosomal rrn operons inactivated but carrying a replaceable multicopy plasmid containing a single rrn operon. The lag time required for growth response on dark/light shift of mutant strains with chromosomal rrnA or rrnB inactivated was increased 50 % over that of the wild-type strain; however, the presence of the plasmid-borne rrn operon restored the lag time. The doubling time of mutant strains carrying only a functional rrnB operon, but not strains carrying only a functional rrnA operon, was significantly longer than that of the wild-type strain. A strain in which essentially all the cellular 23S rRNA contained the mutation C2588A was temperature sensitive at 16 degrees C and 45 degrees C. Position C2588 is equivalent to C2611 of the peptidyltransferase centre in domain V of Escherichia coli 23S rRNA.

Base Sequence↗

PubMed1993

Bioluminescent symbionts of the Caribbean flashlight fish (Kryptophanaron alfredi) have a single rRNA operon.

Ribosomal RNA (rRNA) operon copy number and gene order were determined for the luminous bacterial symbiont of Kryptophanaron alfredi, an anomalopid (flashlight) fish, and estimated for the luminous symbionts of 3 other fish families and of 3 luminous seawater isolates. Compared with the seawater isolates and other fish symbionts, the copy number of rRNA genes in the K. alfredi symbiont was radically reduced, although gene order appeared conserved among all the strains. The K. alfredi symbiont possesses only a single rRNA operon, whereas the other strains examined have minimum copy numbers ranging from 8 to 11. No difference in copy number was observed between light organ and seawater isolates of the same species, or between isolates of the same species from the light organs of 2 different host families. Thus, the anomalopid symbiosis appears unique among characterized light organ symbioses.

Animals↗

PubMed1988

Analysis of transcription and processing signals in the 5' regions of the two Mycoplasma capricolum rRNA operons.

The transcription and RNA processing signals of the rRNA operons (rrnA and rrnB) of Mycoplasma capricolum were analyzed by mapping the 5' ends of in vivo and in vitro synthesized RNAs. The results of both in vitro and in vivo analyses point to the rrnA operon being transcribed from two promoters (P1 and P2) into large precursor RNAs. Transcripts initiating at P1 contain two tRNAs, and probably 16 S, 23 S, and 5 S rRNAs, whereas the transcripts starting from P2 consist only of the three rRNAs. The precursor RNAs are processed via distinct intermediates into mature tRNAs and rRNAs. In vivo experiments indicated that the rrnB operon is transcribed only from one promoter, although a second promoter could be identified using cell free extracts. The rrnB operon does not contain tRNA genes, but the precursor is still processed in the same way as the rrnA precursor that is synthesized from P2.

Base Sequence↗

PubMed1991

Archaeal rRNA operons.

Ribosomal RNA (rRNA) operons of the archaea reflect both the unity and the diversity of this third primary taxon. They have proven to be a rich source of both molecular biological and phylogenetic information.

Archaea↗

PubMed2004

Comparative analysis of the four rRNA operons in Finegoldia magna ATCC29328.

There are four rRNA operons rrnA, rrnB, rrnC and rrnD on the genome of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC29328, which, in contrast to those of Clostridia, are dispersed around the chromosome. Using a BAC library we determined the nucleotide sequences and structures of all four operons, including their flanking regions, and performed comparative analyses. We identified putative boxA sequences in the operons, which should be required for rRNA transcription antitermination, as well as their respective tandem promoters, AT-rich UP elements in the upstream region and Rho-independent terminators in the downstream region. The mosaic features of the operons were revealed. Multiple tRNAs were identified in the downstream region of two operons, 18 in rrnC and 11 in rrnD. They were presumed to form transcription units together with rRNAs. rrnA and rrnB had repeat units with Rho-independent terminators instead of tRNAs in the downstream region. rrnB and rrnC were the most similar in rrn upstream promoter region. Focusing on the sequence variations of rRNA genes, rrnB alone was heterogeneous. In light of previous reports, we also assessed the correlation between intercistronic rRNA sequence differences and distances between the operons, but no positive correlation was seen in this strain.

Base Sequence↗

PubMed2003

First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome.

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.

Acholeplasmataceae↗

PubMed1996

Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons.

The so-called Mycoplasma mycoides cluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of the M. mycoides cluster have two rRNA operons (rrnA and rrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of the M. mycoides cluster, were determined by direct automated solid-phase DNA sequencing. The sequences of both rRNA operons were determined by a novel strategy involving in vitro amplification by PCR with one operon-specific primer pair and one general primer pair. Interestingly, sequence differences (polymorphisms) between the two operons were observed for all strains. Two strains of M. capricolum subsp. capripneumoniae were sequenced, and 15 polymorphisms were found in the type strain (F38) and 17 polymorphisms were found in the other strain (4/2LC). Eight polymorphisms were found in the 16S rRNA genes of the M. mycoides subsp. mycoides small-colony type, and sequence length variations in a poly(A) region were observed in the 16S rRNA genes of the two operons of this species. Secondary-structure analysis showed that polymorphisms were present in both stem and loop regions. The nucleotide substitutions in the polymorphic sites of the stem regions often resulted in a change from a canonical to a noncanonical base pairing or vice versa. A compensatory mutation was never observed in the other nucleotide of the base pair. Phylogenetic analysis based on the 16S rRNA sequences indicated that Mycoplasma sp. strain PG50 should be included in the M. capricolum species group. Furthermore, the 16S rRNA sequences of M. mycoides subsp. capri and the M. mycoides subsp. mycoides large-colony type were 99.9% identical. We therefore suggest that these species be reclassified in a common species group (for instance, "Mycoplasma capri") distinct from the M. mycoides subsp. mycoides small-colony type, which formed an intermediate branch between the M. capricolum species group and the M. capri species group.

Base Sequence↗

PubMed2001

Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens.

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.

Base Sequence↗

PubMed1997

Lack of polymorphism within the rRNA operons of group A streptococci.

Polymerase chain reaction (PCR) ribotyping of many bacterial species has shown that polymorphism of the ribosomal RNA (rRNA) operons, within and between strains, is common. Restriction fragment length polymorphism (RFLP) analysis of the rRNA operons of thirty-two genetically and geographically distinct strains of group A streptococci (GAS) revealed that there are only two major HaeIII PCR-ribotypes. This variation is due to a single nucleotide change within the 16S-23S intergenic spacer regions of these operons. As in many other bacterial species, this spacer region in streptococci also contains the gene for tRNA(ala). Within each GAS isolate, hybridization results are consistent with the presence of six rRNA operons. Interestingly, for a given strain, irrespective of its origin, all six rRNA operons have the same RFLP pattern. This contrasts with the findings in many other bacteria species, where heterogeneity of the rRNA operons within a genome is a common feature. This lack of heterogeneity of rRNA operons in an organism that is known to acquire genetic sequences through horizontal transfer is intriguing.

Base Sequence↗

PubMed1983

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.

Bacillus subtilis↗

PubMed2000

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

Actinomycetales↗

PubMed2002

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups.

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.

Bacillus subtilis↗

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↗