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Results for “rRNA Operon”

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At least 181 records · Page 10Linked to original sources

PubMed1989

Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.

Blotting, Southern↗

PubMed1994

Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing.

Automated solid-phase DNA sequencing was used for determination of partial 16S ribosomal DNA sequences of mycoplasmas. The sequence information was used to establish phylogenetic relationships of 11 different mycoplasmas whose 16S rRNA sequences had not been determined earlier. A biotinylated fragment corresponding to positions 344 to 939 in the Escherichia coli sequence was generated by PCR. The PCR product was immobilized onto streptavidin-coated paramagnetic beads, and direct sequencing was performed in both directions. One previously unclassified avian mycoplasma was found to belong to the Mycoplasma lipophilum cluster of the hominis group. Microheterogeneities were discovered in the rRNA operons of Mycoplasma mycoides subsp. mycoides (SC type), confirming the existence of two different rRNA operons. The 16S rRNA sequence of M. mycoides subsp. capri was identical to that of M. mycoides subsp. mycoides (type SC), except that no microheterogeneities were revealed. Furthermore, automated solid-phase DNA sequencing was used to identify a mycoplasmal contamination of a cell culture as Mycoplasma hyorhinis, which proved to be very difficult by conventional methods. The results suggest that the direct solid-phase DNA sequencing procedure is a powerful tool for identification of mycoplasmas and is also useful in taxonomic studies.

Base Sequence↗

PubMed2001

rrndb: the Ribosomal RNA Operon Copy Number Database.

The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.

Databases, Factual↗

PubMed2002

rRNA promoter activity in the fast-growing bacterium Vibrio natriegens.

The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.

Bacterial Proteins↗

PubMed1987

Gene organization, transcription signals and processing of the single ribosomal RNA operon of the archaebacterium Thermoproteus tenax.

The single ribosomal RNA (rRNA) operon from the extreme thermophile and archaebacterium Thermoproteus tenax was sequenced. Sites of transcriptional initiation and termination were established and the processing sites on the primary transcript were mapped with nuclease S1. The operon contained genes coding for 16S and 23S RNAs but lacked those coding for tRNA and 5S RNA. Transcription initiates 175 bp upstream from the start of the 16S RNA gene (Wich et al., EMBO J. 6, 523-528, 1987) and terminates 49 bp downstream from the 23S RNA gene within a long pyrimidine sequence. An open reading frame downstream from the rRNA operon is transcribed. The sequences bordering both 16S and 23S RNA genes can form putative processing stems in the primary transcript that involve the whole of the 16S-23S RNA spacer. The stems contain irregular features that constitute processing signals and are conserved in other archaebacteria. The 16S RNA stem is cut prior to that of the 23S RNA and RNA maturation follows. An unusual 14 bp helix can form between the extremities of the transcript such that the whole transcript is highly structured and a fork-like structure is formed together with the processing stems. The 23S RNA sequence was aligned with other available 23S-like RNA sequences (Leffers et al., J. Mol. Biol. 195, in press): a putative secondary structure exhibiting archaebacterial-specific features was deduced using comparative sequence analyses. A rooted phylogenetic tree was also derived for the archaebacteria that confirms their division into three major subgroups.

Archaea↗

PubMed1977

Identification of spacer tRNA genes in individual ribosomal RNA transcription units of Escherichia coli.

Transfer RNA genes ("spacer tRNA genes") are present in the spacer region between 16S and 23S rRNA genes in Escherichia coli. We have analyzed spacer tRNA genes carried by seven rRNA operons with different chromosomal locations. Six of these were isolated on plasmids and one on a transducing phage. We found that, in addition to the two previously identified genes for tRNA2Glu and tRNAIIle, there is a spacer tRNA gene which codes for tRNAIBAla. Of the seven rRNA operons studied, three had both tRNAIBAla and tRNAIIle genes, and the remaining four had the tRNA2Glu gene in their spacers. In addition, genes for tRNAIAsp were found near the distal ends of two different rRNA operons.

Alanine↗

PubMed2003

Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants.

Expression of the plastid rRNA operon (rrn) during development is highly regulated at the level of transcription. The plastid rrn operon in most higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a sigma(70)-type promoter with conserved -10 and -35 core promoter elements. To identify functionally important sequences, the tobacco PrrnP1 was dissected in vivo and in vitro. Based on in vivo deletion analysis, sequences upstream of nucleotide -83 do not significantly contribute to promoter function. The in vitro analyses identified an essential hexameric sequence upstream of the -35 element (GTGGGA; the rRNA operon upstream activator [RUA]) that is conserved in monocot and dicot species and suggested that the -10 element plays only a limited role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, the characteristic of strong promoters in prokaryotes. We propose that sigma interaction with the -10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein-DNA) interaction or by protein-protein interaction between the PEP and an RUA binding transcription factor.

Base Sequence↗

PubMed1990

Identification and structural analysis of a ribosomal RNA gene promoter from Thiobacillus ferrooxidans.

The 5'-terminus of a rRNA operon (rrnT2) from Thiobacillus ferrooxidans was characterized. The rRNA promoters from this microorganism were identified by means of a functional assay in Escherichia coli. DNA sequencing of the promoter region, upstream the 16 S rRNA gene, showed the presence of a consensus sequence for bacterial ribosomal promoters. Other features such as a 'discriminator' sequence, antiterminator elements and an upstream hexanucleotide common to several rRNA operons were also found. Two other putative transcription promoters were also identified.

Amino Acid Sequence↗

PubMed2004

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR.

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.

Bifidobacterium↗

PubMed1993

Physiological and genetic regulation of rRNA synthesis in Lactococcus.

The macromolecular composition of Lactococcus was regulated by growth rate in the same general way as that of less fastidious bacteria such as Escherichia coli and Salmonella typhimurium. The ratios of RNA:DNA and RNA:protein increased approximately threefold over a 13.5-fold increase in growth rate, whereas the ratio of DNA:protein remained approximately constant. Using reporter genes fused to a DNA fragment of a cloned lactococcal rRNA operon, promoter activity was located upstream of the 16S rRNA structural gene. This DNA fragment had some characteristics typical of a rrn promoter in E. coli. Two consensus promoter sequences P1 and P2 were located 296 and 157 bp, respectively, upstream of the start of the 16S rRNA gene. Between P2 and the start of the 16S rRNA gene, sequences were identified with typical anti-termination motifs characteristic of E. coli rrn promoter regions. A putative transcription terminator sequence was identified downstream of the 5S rRNA gene and putative primary RNA transcript processing sites at both ends of the lactococcal rRNA operon were also noted.

Amino Acid Sequence↗

PubMed2003

Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

Anti-Bacterial Agents↗

PubMed1991

rRNA transcription rate in Escherichia coli.

The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s.

DNA-Directed RNA Polymerases↗

PubMed1979

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage.

Transducing phage lambdailv5 carries genes for rRNA's, spacer tRNA's (tRNA1 Ile and tRNA1B Ala), and two other tRNA's (TRNA1 Asp and tRNA Trp). We have isolated a mutant of lambdailv5, lambdailv5su7, which carries an amber suppressor mutation in the tRNA Trp gene. A series of deletion mutants were isolated from the lambdailv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion (E. A. Morgan, T. Ikemura, L. Lindahl, A. M. Fallon, and M. Nomura, Cell 13:335--344, 1978) that the genes for tRNA1 Asp and tRNA Trp located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambdailv5su7 transducing phage.

Coliphages↗

PubMed2001

Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance.

Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL(3+), and M. smegmatis rrnB rpsL(3+). M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL(3+) is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL(3+) carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL(3+). Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G-->C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.

Alleles↗

PubMed2002

rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira.

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

Base Sequence↗

PubMed1987

Effects of deletions in the spacer region of the rrnB operon on the transcription of the large ribosomal RNAs from Escherichia coli.

A series of deletions was constructed within the spacer region of the genes for the 16S and 23S RNA on plasmids bearing the rrnB operon. The accumulation and synthesis rates for the 16S and 23S RNAs were determined from normal growing cells and maxicells after transformation with the mutated plasmids. A marked difference in the transcription efficiency of the plasmid-encoded ribosomal 16S and 23S RNAs was observed with cells carrying plasmids, where a sequence motif analogous to the antitermination recognition sequence (Box A) had been deleted. The overall synthesis rate of ribosomal RNAs of such cells was not altered, however, indicating that the difference in transcription rates from the plasmid genes is compensated by altered transcription rates of the corresponding chromosomal genes. In addition, the accumulation of various tRNA species encoded on rRNA operons and non rRNA operons was quantitated and compared. From these results we infer that the regulation of ribosomal RNA transcription does not only occur at the promoter sites but sequence regions possibly involved in antitermination within the operon are crucial for a coordinated synthesis of all ribosomal RNAs.

Chromosome Deletion↗

PubMed1995

Cloning and identification of the hemG gene encoding protoporphyrinogen oxidase (PPO) of Escherichia coli K-12.

Cells of the VSR751 strain, which was previously isolated as a photoresistant revertant of the visA-deleted (hemH-deleted) strain of Escherichia coli K-12, accumulated uroporphyrin (uro), coproporphyrin (copro) and protoporphyrin IX (proto), but did not accumulate as much protoporphyrin as cells of the parental strain (hemH-deleted). Therefore, we concluded that strain VSR751 must be defective in protoporphyrinogen oxidase (PPO), the product of the hemG gene. By complementation analysis using VSR751, we isolated and identified this gene. The hemG gene is located at 86 mim on the E. coli chromosome, just upstream of the rrnA operon, and is transcribed clockwise in the same direction as the rrnA operon. This gene encodes a 181-amino acid protein with a calculated molecular mass of about 21 kDa. Sequence analysis revealed the presence of flavodoxin motif, suggesting tha a cofactor of this enzyme is flavin mononucleotide, which is consistent with the previous report that the mammalian PPO had the flavin cofactor.

Amino Acid Sequence↗

PubMed2000

Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action.

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.

Acetamides↗