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PubMed1983

Efficient read-through of Tn9 and IS1 by RNA polymerase molecules that initiate at rRNA promoters.

Transcription and translation are coupled in most Escherichia coli operons. As a consequence, ribosomes must be present on an mRNA molecule while transcription of the mRNA is in progress or else premature termination of transcription may result. This requirement is most clearly manifested when premature nonsense codons result in polarity in multicistronic operons. Polarity can also result from insertions of transposons and insertion sequences. However, since rRNA operons are not translated, some property of these operons must allow transcription to be uncoupled from translation. In this paper we demonstrate that transposon Tn9 and insertion sequence IS1 are nonpolar or incompletely polar in rRNA operons during normal growth. We also show that essentially all expression of rrn sequences distal to IS1 and Tn9 results from transcripts that originate at rRNA promoters. These results suggest either that rRNA operons possess some mechanism which reduces or prevents termination within rRNA operons or that Tn9 and IS1 can be very inefficient at blocking normal transcription. Insertions of Tn10 in rRNA operons are substantially but incompletely polar. We could not determine whether the residual downstream transcription observed results from promoters within Tn10 or from read-through of Tn10. We discuss the meaning of read-through of Tn9 and IS1 and the residual expression of genes downstream from Tn10 with regard to rRNA operon structure and previous experiments in which polarity of transposons or insertion sequences was observed in protein-encoding operons.

DNA Transposable Elements↗

PubMed1992

Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui.

The halophilic archaebacterium, Haloarcula marismortui, contains two nonadjacent ribosomal RNA operons, designated rrnA and rrnB, in its genome. The 16S rRNA genes within these operons are 1472 nucleotides in length and differ by nucleotide substitutions at 74 positions. The substitutions are not uniformly distributed but rather are localized within three domains of 16S rRNA; more than two-thirds of the differences occur within the domain bounded by nucleotides 508 and 823. This domain is known to be important for P site binding of aminoacylated tRNA and for 30-50S subunit association. Using S1 nuclease protection, it has been shown that the 16S rRNAs transcribed from both operons are equally represented in the functional 70S ribosome population. Comparison of these two H. marismortui sequences to the 16S gene sequences from related halophilic genera suggests that (i) in diverging genera, mutational differences in 16S gene sequences are not clustered but rather are more generally distributed throughout the length of the 16S sequence, and (ii) the rrnB sequence, particularly within the 508-823 domain, is more different from the out group sequences than is the rrnA sequence. Several possible explanations for the evolutionary origin and maintenance of this sequence heterogeneity within 16S rRNA of H. marismortui are discussed.

Base Sequence↗

PubMed1999

RecA-Mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA.

Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.

Alleles↗

PubMed2000

Characterization of the 16S rRNA gene V2 region and the rrn operons of Gardnerella vaginalis.

Ribosomal RNA (rRNA) gene polymorphism was apparent when Gardnerella vaginalis DNA restriction profiles were hybridized with nonradioactively labeled total rRNA isolated from this bacterium. In contrast, use of a polymerase chain reaction (PCR)-based 16S rRNA gene V2 region resulted in a 118-bp V2-PCR amplicon that was specific and common in all 30 tested G. vaginalis isolates. In addition to providing a G. vaginalis-specific fingerprint, when the V2-PCR amplicon along with total rRNA were utilized as probes, a partial rRNA gene restriction map could be constructed. G. vaginalis contains two rrn operons with an EcoRI fragment of 1.6 kb common to both.

DNA Fingerprinting↗

PubMed1998

DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates.

To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified by PCR and the PCR products were sequenced. Three isolates had identical 16S rRNA sequences and two isolates had sequences that differed from the others by only one nucleotide.

Base Sequence↗

PubMed2005

The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies.

Ribosomal RNAs in E. coli are transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for the rrnB and rrnD fragments and less pronounced for the rrnA and rrnE operons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.

Bacterial Proteins↗

PubMed1993

Construction of the Bacillus subtilis chromosome physical map and the strategy for mapping newly isolated genes in one membrane filter for hybridization.

A complete physical map of the Bacillus subtilis 168 chromosome was constructed. The merging of this physical map is expected not only to provide important insights into the organization and rearrangement of genes of this species but also to be a powerful means for the genome analysis. One of the most practical aspects is rapid and accurate mapping of newly isolated genes using a single membrane filter for hybridization. This protocol proved that not only unique genes but also multiple homologous genes dispersed on the chromosome can be physically mapped.

Bacillus subtilis↗

PubMed2001

An E. coli 5S rRNA deletion mutant useful for the study of 5S rRNA structure/function relationships.

We report on the construction of a novel strain of E. coli that can be useful for studies on the structure/function relationship of 5S rRNAs. The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA. The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function. We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence.

Escherichia coli↗

PubMed2004

Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach.

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 degrees C, and Escherichia coli B/r, grown at 37 degrees C. A frame of reference was established based on quantitative relationships observed between specific growth rates ( micro ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally ( micro =0.30 h(-1)) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli ( micro =1.73 h(-1)); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating micro to the number of rrn operons per genome. Values of micro =0.69 h(-1) and micro =1.00 h(-1) were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

Bacterial Proteins↗

PubMed1994

Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR.

Mycoplasma sp. (strain F38) is the causative agent of contagious caprine pleuropneumonia, which is a goat disease of great global concern. Strain F38 belongs to the so-called "Mycoplasma mycoides cluster," and the members of this cluster have many biochemical and serological properties in common, which makes it difficult to differentiate between them by conventional methods. Their phylogenetic interrelationship are thus uncertain. The 16S rRNA gene of the rrnB operon from strain F38 was cloned and sequenced. The sequence was compared with the 16S rRNA sequences of related mycoplasmas, and phylogenetic trees were constructed by parsimony analysis. A three-way ambiguity among strain F38, Mycoplasma capricolum, and Mycoplasma sp. strain PG50 was observed in the trees. This observation is in agreement with a recent proposal to reclassify strain F38 and M. capricolum. A primer set was designed for in vitro amplification by PCR of a fragment of the 16S rRNA genes from the M. mycoides cluster. The amplimers of strain F38 could be distinguished easily from the corresponding amplimers from other members of the M. mycoides cluster by restriction enzyme analysis with PstI. This observation was utilized to design an identification system for strain F38. Part of the 16S rRNA gene of the rrnA operon from strain F38 was also cloned, and several sequence differences between the two rRNA operons were discovered, revealing microheterogeneity between the two 16S rRNA genes of this organism.

Acholeplasma↗

PubMed1998

Specific PCR primers from the 16S-23S rRNA spacer region for the rapid detection and identification of Actinobacillus seminis.

Actinobacillus seminis is a common cause of ovine epididymitis and ram infertility. The ability to detect and identify this organism promptly is important commercially for the quality control of ram semen samples. Actinobacillus seminis is a fastidious and slow-growing bacterium and primary isolation and presumptive identification can be difficult and time-consuming. In this study, two ribosomal operons, termed rrnA and rrnB, have been characterized in the A. seminis genome, and these contain one and two tRNAs, respectively, in the spacer region between the 16S and 23S rRNA genes. Species-specific primers for A. seminis were developed from the sequence of the spacer region of rrnB for the identification and detection of A. seminis by PCR. The PCR assay was specific for A. seminis and gave no amplification products with phenotypically similar organisms such as Histophilus ovis. Storage solution used to preserve semen for long-term storage was found to inhibit the PCR. Therefore, for diagnostic purposes, the assay would best be performed after primary isolation or perhaps on fresh semen prior to storage if obvious contamination is indicated.

Actinobacillus↗

PubMed2003

The number and organization of the rRNA genes of several strains of Mycobacterium simiae.

The type strain of Mycobacterium simiae and four Cuban strains, each representing a group of variants sharing a characteristic pattern of glycopeptidolipids, were investigated. Each of the five strains was found to have a single rRNA (rrn) operon per genome. Each rrn operon was found to be located downstream from murA. Unusually for slow-growing mycobacteria, three transcription start points were identified for each operon. Gene sequences were established extending from near to the 3'-ends of murA, the intergenic regions and the 5'-ends of the 16S rDNAs. Characteristic strain differences were identified.

Base Sequence↗

PubMed1992

The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA.

Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.

Base Sequence↗

PubMed1981

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

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

Base Sequence↗

PubMed2005

Molecular phylogenetic analysis of Vibrio cholerae O1 El Tor strains isolated before, during and after the O 139 outbreak based on the inter-genomic heterogeneity of the 16S-23S rRNA intergenic spacer regions.

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.

Cloning, Molecular↗

PubMed1997

Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo.

A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription. In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis. Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis. Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis. In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium. This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA. We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.

Escherichia coli↗

PubMed1991

A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA.

S. coelicolor A3(2) contains six ribosomal RNA operons. Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons. Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon-containing bands. The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical. Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD. Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region.

Amino Acid Sequence↗

PubMed1988

Control of the tRNA-tufB operon in Escherichia coli. 1. rRNA gene dosage effects and growth-rate-dependent regulation.

'Ribosome feedback' effects on the expression of the genes specifying tRNA and EF-Tu in E. coli have been studied at increased rrnB doses (rRNA gene doses). We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRNA-tufB operon, but leaves the 5S rRNA content unaffected. Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF-Tu that can be fully accounted for by a decrease in EF-TuB level. The drop in EF-TuB content (approx. 25%) is much smaller than that in tRNA content (approx. 80%). The synthesis rate of total EF-Tu is hardly affected, indicating that the turnover of EF-Tu has not changed. The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage. Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent. The relatively small drop in EF-Tu synthesis must be due, therefore, to an enhancement of the number of EF-Tu molecules synthesized per mRNA molecule. Apparently a post-transcriptional mechanism, regulating EF-Tu synthesis, becomes operative under these conditions. Growth-rate-dependent regulation of the tRNA-tufB operon has been studied using lysogens carrying tRNA':lacZ and tRNA-tufB':lacZ operon fusions and a tufB':lacZ' gene fusion. These experiments show that the cellular contents of tRNA, tufB RNA and EF-TuB vary in direct proportion to the growth rate. This indicates that growth rate control of tRNA-tufB operon transcription resembles that of stable RNA operons and not of r-protein operons, and that the read-through of the terminator at the end of the tRNA gene cluster remains unaltered.

Escherichia coli↗