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PubMed1998

A light-activated DNA-binding factor stimulates transcription of the rrnA operon in the cyanobacterium Synechococcus sp. PCC 6301.

In Synechococcus sp. strain PCC 6301, ribosomal RNA (rRNA) synthesis occurs at specific times during the growth cycle in the light. When light-grown cultures are placed in the dark, rRNA synthesis and cell division stop abruptly. It is shown here that a partially purified DNA-binding protein binds downstream of the rRNA operon (rrnA) P1 promoter in the light but not in the dark. When the DNA binding protein is added to in vitro transcription assays, run-off transcripts are produced in the light but not, under dark conditions. The results indicate that a light-activated regulatory molecule is involved in stimulating rRNA synthesis during the normal cell growth cycle of Synechococcus in the light.

Binding Sites↗

PubMed1995

Complete sequences and organization of the rrnA operon from campylobacter jejuni TGH9011 (ATCC43431).

The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion. rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3' prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA(Ala) and tRNA(Ile), were present in the order 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', which is opposite of the typical 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-3' structure observed in other bacteria.

Base Sequence↗

PubMed1999

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.

Base Sequence↗

PubMed1998

Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui.

The genome of the halophilic archaeon Haloarcula marismortui contains two rRNA operons designated rrnA and rrnB. Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized. The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated. The two operons exhibit other profound differences that include (i) the presence in rrnA and the absence in rrnB of tRNAAla and tRNACys genes in the intergenic and distal regions, respectively, (ii) divergent 5' flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA from rrnA operon transcripts and of 23S rRNA from rrnB operon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA from rrnB operon transcripts follows an unusual and different pathway that is apparently devoid of any 5' processing intermediate.

Base Sequence↗

PubMed1995

rRNA operon restriction derived taxa for Streptomyces (RiDiTS).

A method for grouping Streptomyces strains by fingerprints of their rRNA operons is described. In polyacrylamide gels, multicopy rRNA operon fragments in Streptomyces genomic MseI fingerprints produced intense bands which are well resolved from the less conspicuous low copy fragments interspersed between them. The high intensity multicopy rRNA bands are easily distinguished from the low intensity bands, eliminating the need for Southern blot hybridization to visualize the rRNA fragments. Direct evidence that the high-intensity bands in these polyacrylamide gels originated from rRNA operons was provided by a 'differential' Southern blot technique. We have used this method to assign 98 strains to 11 rRNA fingerprint type groups. This clustering method may be applicable to any prokaryote with a high G+C content genome.

Blotting, Southern↗

PubMed1988

Promoter of the Mycoplasma pneumoniae rRNA operon.

RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene. The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132. It was concluded that transcription of the rRNA operon of M. pneumoniae is initiated by a single promoter. The nucleotide sequence of the region is presented.

Base Sequence↗

PubMed2002

Characterization of an rRNA operon (rrnB) of Mycobacterium fortuitum and other mycobacterial species: implications for the classification of mycobacteria.

Mycobacteria are thought to have either one or two rRNA operons per genome. All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene. We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3' end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase. The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5' 3-mag-tyrS-rrnB 3'. The rrnB operon of M. fortuitum was found to have a single dedicated promoter. During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis. Genomic DNA was isolated from eight other fast-growing mycobacterial species. Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences. The results revealed that both rrnA and rrnB operons were present in each species. The results form the basis for a proposed new scheme for the classification of mycobacteria. The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences.

Base Sequence↗

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↗

PubMed1984

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.

Base Sequence↗

PubMed1983

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.

Base Sequence↗

PubMed1993

Identification of cis-acting regulatory regions upstream of the rRNA operons of Rhodobacter sphaeroides.

The promoter region(s) for the rRNA operons of Rhodobacter sphaeroides was identified. By utilizing both in vivo and in vitro techniques, the transcriptional start sites of all three operons were identified. Upstream of the transcriptional start, -10 and -35 promoter regions that bear little similarity to typical Escherichia coli promoter sequences were identified. In addition to the promoter sequences, probable Fis protein-binding sites were identified upstream of all three rRNA operons. Transcriptional fusions of the promoter regions from rrnA and rrnB were constructed by utilizing the reporter molecule encoded by xylE and analyzed under various growth conditions, in both a wild-type background and an rrnBC mutant background. Production of the xylE gene product (catechol 2,3-dioxygenase) was always greatest under photosynthetic growth conditions. However, the upstream region of rrnB, when fused with xylE, produced significantly more catechol 2,3-dioxygenase than did analogous regions of rrnA, suggesting that the promoters of the rrn operons differ in strength. These results were further confirmed by the study of mutant strains altered for the rrn operons either singly or in combination. Little or no expression of the xylE gene was manifest in E. coli when directed by rDNA sequences derived from R. sphaeroides.

Base Sequence↗

PubMed1978

Some rRNA operons in E. coli have tRNA genes at their distal ends.

We have previously isolated seven rRNA operons on plasmids or lambda transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNAGlu2 or both tRNAIle1 and tRNAAla1B genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNATrp and tRNAAsp1 are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNATrp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNATrp and tRNAAsp1 at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNAGlu2, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.

Chromosome Mapping↗

PubMed1982

Structural and functional analysis of an rRNA operon and its flanking tRNA genes from Zea mays chloroplasts.

The complete analysis of an rRNA operon from Zea mays chloroplasts and its comparison with other plastidic or bacterial rRNA operons is presented. The maize operon contains structural genes for 16S, 23S and 4.5S rRNA species, a leader region proximal to the 16S rRNA gene and a 2.4 kb spacer between the 16S and 23S rRNA genes. Within the spacer DNA sequence are two tRNA genes which code for a tRNAIle and tRNAAla species but each gene is split by large intervening sequences of 949 and 806 bp, respectively. 4.5S rRNA is a structural equivalent of the 3' terminal region of bacterial 23S rRNA. The operon is flanked at its 5' side by a tRNAVal gene and at its 3' side by a 5S rRNA gene. Both these genes are probably not included in the large, primary precursor rRNA transcript.

Base Sequence↗

PubMed1999

Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons.

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

Chromosomes, Bacterial↗

PubMed1987

Chlamydial rRNA operons: gene organization and identification of putative tandem promoters.

We isolated and characterized the rRNA operons of murine Chlamydia trachomatis. By exhaustively screening a library of chlamydial DNA and by blot hybridization of genomic DNA, we showed that there are only two rRNA operons in C. trachomatis. S1 nuclease protection and primer extension analysis were used to map the 5' and 3' ends of the mature 16S and 23S transcripts in both rRNA cistrons and, additionally, to demonstrate the lack of intervening sequences in these genes. The 5' ends of the presumed primary rRNA transcript were located and found to originate at two tandem sites separated by 100 base pairs. The two tandem chlamydial rDNA transcripts were not differentially regulated. Their products were coordinately expressed and were detectable as early as 9 h postinfection. However, the upstream transcript was only 10% as abundant as the downstream transcript. The sequences surrounding the transcription initiation sites bore little homology with each other or with the classic Escherichia coli -10 and -35 promoter sequences. This finding suggests that chlamydial transcription signals may differ from those of previously studied procaryotes.

Base Sequence↗

PubMed1991

Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens.

All 10 rRNA operons have been situated on the genome map of the anaerobic pathogen Clostridium perfringens. Four of these have been cloned and partially sequenced, and their transcriptional patterns in vivo and in vitro have been examined. Expression of rrnA, rrnB, and rrnE is directed by tandem promoters, P1 and P2, whereas rrnH is the only one to be expressed from a single promoter, which resembles P1. On inspection of the nucleotide sequences of the control regions, several sites which might be involved in the regulation of rrn expression were identified. These include a possible upstream activating region which could be recognized by the C. perfringens equivalent of the Escherichia coli Fis protein and a stringent response target site. Studies of maturation of 16S RNA identified two 5' cleavage sites and sequence analysis showed the dG+dC content of its gene, rrs, to be 52%, which is twice that of the genome.

Amino Acid Sequence↗

PubMed2004

Two distinct types of rRNA operons in the Bacillus cereus group.

The Bacillus cereus group includes insecticidal bacteria (B. thuringiensis), food-borne pathogens (B. cereus and B. weihenstephanensis) and B. anthracis, the causative agent of anthrax. The precise number of rRNA operons in 12 strains of the B. cereus group was determined. Most of the tested strains possess 13 operons and the tested psychrotolerant strains contain 14 operons, the highest number ever found in bacteria. The separate clustering of the tested psychrotolerant strains was confirmed by partial sequencing of several genes distributed over the chromosomes. Analysis of regions downstream of the 23S rRNA genes in the type strain B. cereus ATCC 14579 indicates that the rRNA operons can be divided into two classes, I and II, consisting respectively of eight and five operons. Class II operons exhibit multiple tRNA genes downstream of the 5S rRNA gene and a putative promoter sequence in the 23S-5S intergenic region, suggesting that 5S rRNA and the downstream tRNA genes can be transcribed independently of the 16S and 23S genes. Similar observations were made in the recently sequenced genome of B. anthracis strain Ames. The existence of these distinct types of rRNA operons suggests an unknown mechanism for regulation of rRNA and tRNA synthesis potentially related to the pool of amino acids available for protein synthesis.

Bacillus cereus↗

PubMed1988

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.

Anti-Bacterial Agents↗