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PubMed2004

Control of rRNA synthesis in Escherichia coli: a systems biology approach.

The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (V(max)/K(m)) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation.

Escherichia coli↗
PubMed1996

Sex chromosome linkage of 5S rDNA in rainbow trout (Oncorhynchus mykiss).

The karyotype of the rainbow trout is characterized by a primitive XX/XY sex-determining chromosomal system. (Thorgaard et al., 1977). In the present study using FISH we have physically linked the 5S rRNA genes to the partially undifferentiated X chromosome pair. PCR amplified 5S rDNA was used for FISH and hybridization signals indicated that the genes were duplicated, present in one acrocentric and one metacentric pair of chromosomes. After analyzing several individuals, the female metaphases showed four fluorescent signals whereas males presented only three signals. Two of the three signals obtained in males corresponded to the metacentric pair whereas the single signal was mapped to the heterochromatin that cytologically differentiates the X chromosome from the Y chromosome. Double FISH experiments demonstrated that the 5S rDNA which is not sex linked is located at the NOR bearing arm close to the major ribosomal RNA genes (5.8S, 18S and 28S), similar to the situation observed in Atlantic salmon (Pendas et al., 1994a).

Animals↗
PubMed1997

Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos.

When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.

Animals↗
PubMed1999

Molecular characterization of Streptococcus equi subspecies equi isolated from an Ethiopian camel by ribotyping and PCR-ribotyping.

The aim of this study was to characterize a S. equi subspecies equi strain isolated from an Ethiopian camel by different molecular techniques (Ribotyping and PCR-Ribotyping). We compared the results obtained with those generated from two strains of the Pasteur Collection. The ribotyping showed the highest power of differentiation, distinguishing between the strains analyzed, whereas PCR-Ribotyping was able only to differentiate the camel isolate but not the strains from the Pasteur Collection. The application of this technique will be very useful to establish a clonal relationship among equine and camelids strains and help the prevention and cure of the equine and camel pathology.

Animals↗
PubMed1992

DNA melting within stable closed complexes at the Escherichia coli rrnB P1 promoter.

Several transcription complexes are shown to form on the E. coli ribosomal rrnB P1 promoter in vitro. These include two closed complexes that are sensitive to heparin attack, and one open complex. The closed complexes are unusual in that they are both highly specific and stable, properties associated with the atypical DNA sequence of this promoter. The effector ppGpp does not prevent closed complex formation but does reduce the level of open complexes that form.

Adenosine Triphosphate↗
PubMed1992

Synthesis of cRNA probes from PCR-generated DNA.

We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.

Animals↗
PubMed1990

[Role of SV40 T antigen binding sites within the 5' flanking region of human U1 and U2 snRNA genes].

The 5' flanking regions of the genes encoding human U1 and U2 snRNA each contain multiple copies of consensus pentanucleotide 5' GA/GGGC 3' which bind specifically to the SV40 T antigen. To examine the role of T antigen binding sites, the mutants deleted and substituted the pentanucleotide sequences from 5' flanking of U1 and U2 genes were constructed by oligonucleotide-directed DNA synthesis. A similar transcription level was observed between these mutants and wild type in cell-free transcription system. Both U1 and U2 snRNA synthesized in vitro initiate from upstream of the cap site identified in vivo. This imply that deletion and substitution of T antigen binding sites unaffect the transcriptional property of U1 and U2 genes in vitro. We have shown that deletion mutants virtually eliminated U1 and U2 snRNA synthesis in HeLa, 293 and oocytes cells, while substitution mutants fully restored transcription to wild type level. Thus, while the region located between -11 and -42 of human U1 or -58 and -90 of human U2 DNA is required for transcription in these cells, the wild type DNA sequence in this region is not a prerequisite for transcription. This suggests that this region is required for DNA spacing rather than nucleotide sequence.

Animals↗
PubMed1994

Genomic relatedness among reference strains of different Streptococcus suis serotypes.

Hybridization studies using genomic DNA and a rDNA probe revealed genetic relatedness among reference strains of different Streptococcus suis serotypes. Although most serotype 22 isolates are biochemically atypical, the reference strain of capsular type 22 is genetically related to other S. suis serotypes, but not to Streptococcus pneumoniae. Using DNA digested with BamHI and BglII for ribotyping, some S. suis reference strains had common patterns, but this analysis mainly revealed variations in patterns of S. suis strains of different serotypes.

Blotting, Southern↗
PubMed1994

[Comparative study of single strand conformation polymorphism of 4.5S RNA gene in enterobacteria].

A recently developed technique, non-isotopic single strand conformation polymorphism analysis (PCR-SSCP), was applied to study the conserved feature of 4.5S RNA gene in enterobacteria. The 4.5S RNA gene was amplified by the polymerase chain reaction, using the template DNA extracted respectively from five strains of Escherichia coli and three strains of different genera in Enterobacteriaceae, i.e. Proteus vulgaris, Serratia marcescens and Enterobacter aerogenes. The PCR products were then carried out SSCP analysis. The experimental results showed that there seemed to be no detectable differences in the size and single strand conformation of 4.5S RNA genes from above strains, except the negative strand conformation of Enterobacter aerogenes. Thus it can be seen that the secondary structures of 4.5S RNA gene in enterobacteria are quite conservative.

Base Sequence↗
PubMed1993

The kinetics of formation of complexes between Escherichia coli RNA polymerase and the rrnB P1 and P2 promoters of Bacillus subtilis. Effects of guanosine tetraphosphate on select steps of transcription initiation.

The kinetics of formation and dissociation of heparin-resistant transcription initiation complexes between Escherichia coli RNA polymerase and the rrnB P1 and P2 promoters from Bacillus subtilis were investigated using a gel retardation assay. The results suggest that the formation of polymerase-promoter complexes proceeds by a three-step reaction mechanism. The bimolecular collision between free RNA polymerase and the promoter creates a heparin-sensitive complex, which then isomerizes to an initial, and then a subsequent, heparin-resistant complex. We propose that a sequential mechanism best describes the bimolecular collision and that the forward rate constants predominate in the overall rate of heparin-resistant complex formation. At 35 degrees C, the association of polymerase with P1 and P2 was very rapid (ka = 1.5 - 2.1 x 10(8) M-1 s-1; kf > or = 0.56 s-1). Direct information on the formation of the closed and intermediate transcription complexes and indirect information on the formation of open complexes suggest that guanosine tetraphosphate did not differentially affect any step between growth rate-regulated and non-growth rate-regulated rRNA promoters by more than 2-fold.

Bacillus subtilis↗
PubMed1997

Ribosomal RNA gene dosage in marine bacteria.

Ribosomal RNA gene dosage was determined for 20 marine heterotrophic bacteria using short probes (< 600 bp) from the Escherichia coli 16S rRNA gene and Southern blot analysis. All Bacterial strains had between 4 and 10 copies of the 16S rRNA genes in their genomes. This report presents important preliminary data for developing quantitative molecular methods to address population dynamics of marine based of 16S rRNA sequences.

Bacteria↗
PubMed1997

Rapid detection of Mycobacterium tuberculosis in respiratory specimens, blood and other non-respiratory specimens by amplification of rRNA.

SETTING: Diagnostic methods employing gene technology based on amplification of DNA or RNA are expected to improve the speed, sensitivity, and specificity of Mycobacterium tuberculosis detection. The Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) enables the amplification and detection of M. tuberculosis rRNA directly from respiratory specimens. OBJECTIVE: To evaluate the performance of the AMTDT in direct detection of M. tuberculosis in respiratory specimens, blood and other clinical samples, and to compare this method with conventional culture and staining techniques. DESIGN: A total of 554 samples from 450 patients were examined in this study. All clinical specimens (with the exception of bone marrow aspirates and blood samples) were digested and decontaminated with sodium dodecyl (lauryl) sulfate (SDS)-NaOH. Bone marrow aspirates and blood samples were treated with 10% SDS. All processed samples were stained by auramine-rhodamine fluorochrome and inoculated onto Löwenstein-Jensen and Coletsos solid media, and into BACTEC-12B medium. In addition, the blood samples were inoculated into BACTEC 13A medium. The AMTDT was performed according to manufacturer's instructions. In those cases where discrepant results were obtained for AMTDT and cultures, patients' clinical data and other microbiological results were evaluated. RESULTS: The sensitivity, specificity, and positive and negative predictive values for AMTDT were 87.5, 100, 100, and 96.7%, respectively, in respiratory specimens and 86.8, 100, 100, and 92.8%, respectively, in non-respiratory specimens. The differences in sensitivity of these two groups of specimens were not highly statistically significant (P > 0.005). CONCLUSION: The sensitivity and specificity of the AMTDT were satisfactory for detection of M. tuberculosis in all types of clinical samples. Some minor changes in assay format and laboratory protocols may increase the sensitivity of the AMTDT without adversely affecting its specificity.

Bacteriological Techniques↗
PubMed1977

Characterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli.

We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind.

Bacterial Proteins↗
PubMed1993

Some base substitutions in the leader of an Escherichia coli ribosomal RNA operon affect the structure and function of ribosomes. Evidence for a transient scaffold function of the rRNA leader.

Recently, we reported on the creation of a systematic series of C to T transition mutations, located between 19 and 45 nucleotides upstream of the mature 16 S RNA 5' end of a complete, plasmid encoded ribosomal RNA operon. We showed that some of these base transitions have pronounced effects on the growth phenotype of mutant cells, and on the stability of their 16 S RNA as well as on the association capability of their ribosomal subunits. From these observations we concluded that the mutated leader region has post-transcriptional functions in the biogenesis of ribosomes. To further substantiate our conclusions we have now analyzed the growth phenotypes of some leader mutants in more detail, and show here that they are temperature dependent. Furthermore, we have isolated ribosomal RNA, 70 S ribosomes and ribosomal subunits from wild-type and mutant strains and subjected them to a detailed structural and functional analysis. We show that processing and maturation of the 16 S RNA is not altered as a consequence of the base transitions in the leader. In contrast, comparison of the protein composition of wild-type and mutant 30 S particles by two-dimensional gel electrophoresis revealed specific differences. Wild-type 30 S subunits, which are not tightly bound to 50 S, are lacking many ribosomal proteins, while the same fraction of ribosomes from mutant cells has an approximately complete r-protein set, and instead contains some additional non-ribosomal proteins. The translational activity of mutant and wild-type total ribosome preparations was analyzed in vitro. Ribosomes from slowly growing mutants show a significantly reduced in vitro translational activity, which is caused by the 30 S subunits. In contrast, the defects in association reside mainly in the 50 S subunits. Our results demonstrate that some base substitutions in the leader of an Escherichia coli rRNA operon affect the structure and function of ribosomes, although the mutated region is not part of the particles finally formed. This finding implies that at least part of the leader region assists the structure formation of functional 30 S subunits, before it is cut away and discarded. We argue that the rrn leader thus fulfills the functional criteria of a transient molecular scaffold.

Base Sequence↗
PubMed1969

Transcriptional mapping: functional organization of the ribosomal and transfer ribonucleic acid cistrons in the Bacillus subtilis genome.

The transcriptional mapping method developed previously has been used to investigate the functional organization of the ribosomal ribonucleic acid (rRNA) and transfer ribonucleic acid (tRNA) cistrons in the Bacillus subtilis genome. The sensitivities of the syntheses of these RNA species to actinomycin D indicated that (i) large polycistronic groupings of the tRNA cistrons exist, but (ii) the 16S and 23S rRNA cistrons are probably transcribed as individual units, and (iii) each 5S rRNA cistron is part of an operon containing a 23S rRNA cistron.

Bacillus subtilis↗