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Results for “rRNA Operon”

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At least 343 records · Page 19Linked to original sources

PubMed1993

Characterization of the fMet initiator tRNA gene of Bartonella bacilliformis.

A ribosomal RNA operon was cloned from the agent of human Oroya fever, Bartonella bacilliformis. The 3' end of the operon contains a 77-nucleotide (nt) N-formylmethionine (fMet) initiator tRNA-encoding gene with 100% sequence identity to the fMet tRNA-encoding gene of Rhodobacter sphaeroides. The 3' region downstream from the gene has an 8-nt inverted repeat and 28 contiguous direct repeats of the heptamer 5'-TTCTCTA. To date, B. bacilliformis and R. sphaeroides are the only known eubacteria which have an fMet tRNA-encoding gene within the 3' end of their rRNA operons.

Bartonella↗

PubMed1995

Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function.

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.

Base Sequence↗

PubMed2005

Structure of a Mycobacterium tuberculosis NusA-RNA complex.

NusA is a key regulator of bacterial transcriptional elongation, pausing, termination and antitermination, yet relatively little is known about the molecular basis of its activity in these fundamental processes. In Mycobacterium tuberculosis, NusA has been shown to bind with high affinity and specificity to BoxB-BoxA-BoxC antitermination sequences within the leader region of the single ribosomal RNA (rRNA) operon. We have determined high-resolution X-ray structures of a complex of NusA with two short oligo-ribonucleotides derived from the BoxC stem-loop motif and have characterised the interaction of NusA with a variety of RNAs derived from the antitermination region. These structures reveal the RNA bound in an extended conformation to a large interacting surface on both KH domains. Combining structural data with observed spectral and calorimetric changes, we now show that NusA binding destabilises secondary structure within rRNA antitermination sequences and propose a model where NusA functions as a chaperone for nascently forming RNA structures.

Amino Acid Sequence↗

PubMed1993

Genetic diversity within isolates of mutans streptococci recognized by an rRNA gene probe.

A total of 79 clinical isolates of mutans streptococci and five laboratory strains representing serotypes c, d, e, f, and g were genotyped by a nonradioactive hybridization method with the rrnB rRNA operon of the Escherichia coli chromosome as a probe. The hybridization patterns of chromosomal DNA fragments obtained by digestion with restriction endonucleases HindIII, SmaI, and BamHI revealed genotypic heterogeneity among the serotypes and among isolates of the same serotype recovered from unrelated subjects. Diversity also existed among isolates obtained from a single subject. For 5 of 13 subjects studied, two or three genotypes within serotypes were found, while eight subjects harbored the same number of genotypes as serotypes. The data show that the method utilizing the rRNA gene probe is of value in determining the molecular epidemiology of isolates of mutans streptococci.

Escherichia coli↗

PubMed1999

Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients with Pneumocystis carinii pneumonia.

A nested PCR, amplifying a portion of the gene encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) of Pneumocystis carinii sp. f. hominis was applied to oropharyngeal samples obtained on repeated occasions from 12 HIV-infected patients with P. carinii pneumonia (PCP) to monitor response to anti-P. carinii treatment. Genotyping of P. carinii sp. f. hominis was also performed on paired samples of oropharyngeal and broncho-alveolar lavage samples before the start of treatment, and on oropharyngeal samples during the course of treatment, by analysis of sequence variation at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon. When a simple dilutional method was used, a reduction in the amount of amplification product was observed in samples from all patients during the course of treatment. In eight of the 12 patients, a single ITS sequence type was found in the oropharyngeal samples and also in the paired broncho-alveolar lavage sample. A mixed infection was identified in the samples from three patients. In eight patients, the ITS sequence types identified in the oropharyngeal sample were the same as in the broncho-alveolar lavage sample. Nested PCR amplifying the mt LSU rRNA on oropharyngeal samples provides a non-invasive method of monitoring response to treatment of PCP. ITS sequence typing of P. carinii sp. f. hominis from oropharyngeal samples appears to be a reliable alternative to broncho-alveolar lavage samples and provides a non-invasive tool for further epidemiological studies.

AIDS-Related Opportunistic Infections↗

PubMed1999

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Bacteria↗

PubMed2002

NTP-sensing by rRNA promoters in Escherichia coli is direct.

We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.

Adenosine Triphosphate↗

PubMed2001

Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.

Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniae infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

Bacterial Proteins↗

PubMed2005

Characterization of the alternative sigma factor sigmaG in Streptomyces coelicolor A3(2).

Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.

Amino Acid Sequence↗

PubMed1999

Diagnosis of contagious bovine pleuropneumonia by PCR-laser- induced fluorescence and PCR-restriction endonuclease analysis based on the 16S rRNA genes of Mycoplasma mycoides subsp. mycoides SC.

As contagious bovine pleuropneumonia (CBPP) is spreading fast in many African countries, there is an increasing demand for rapid and sensitive diagnostic methods that can be used to confirm the initial diagnosis based on clinical symptoms or pathological findings. Two PCR-based diagnostic systems for identification of the infectious agent, Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC), in various samples are presented. Both systems involve group-specific amplification of the two 16S rRNA genes from mycoplasmas of the M. mycoides cluster. The laser-induced fluorescence assay is based on a unique sequence length difference between the two 16S rRNA genes in M. mycoides SC. This region was amplified by PCR, and the products were separated by polyacrylamide gel electrophoresis in a DNA sequencer. The resulting electropherogram showed two peaks for strains of M. mycoides SC and one peak for all other members of the M. mycoides cluster. The second system was based on restriction endonuclease analysis and agarose gel electrophoresis. Restriction of amplicons from a region containing a polymorphism, which is found in M. mycoides SC only, resulted in an extra band on the agarose gel because an AluI site is lacking in the rrnA operon. Specimens from cows with postmortem signs of CBPP were analyzed with the two PCR systems. M. mycoides SC was clearly identified in pleural fluid and lung tissue, and the methods were found to be robust and rapid. The results were in agreement with those obtained by conventional diagnostic techniques.

Animals↗

PubMed1993

Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions.

To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. difficile strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Typing Techniques↗

PubMed2003

Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics.

Acidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA-based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210,000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co-linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine-DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two-component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well-studied bacterial phyla.

Bacteria↗

PubMed2006

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes.

BACKGROUND: The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA) sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae), in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR) featuring an inverted rRNA operon and a small single-copy (SSC) region containing 14 genes normally found in the large single-copy (LSC) region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. RESULTS: The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of Oltmannsiellopsis cpDNA more closely resembles that of Chlorella (Trebouxiophyceae) cpDNA. CONCLUSION: The chloroplast genome of the last common ancestor of Oltmannsiellopsis and Pseudendoclonium contained a minimum of 108 genes, carried only a few group I introns, and featured a distinctive quadripartite architecture. Numerous changes were experienced by the chloroplast genome in the lineages leading to Oltmannsiellopsis and Pseudendoclonium. Our comparative analyses of chlorophyte cpDNAs support the notion that the Ulvophyceae is sister to the Chlorophyceae.

Base Sequence↗

PubMed1999

Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation.

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.

Bacterial Proteins↗

PubMed2001

Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts.

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.

DNA, Fungal↗

PubMed1999

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.

Animals↗

PubMed1987

Novel expression of the ribosomal RNA genes in the extreme thermophile and archaebacterium Desulfurococcus mobilis.

The single 16S/23S rRNA operon of the sulfur-dependent extreme thermophile Desulfurococcus mobilis is transcriptionally uncoupled from the single 5S RNA gene. Transcription of the former operon initiates at five sites, P5-P1. RNA polymerases intiating at sites P5-P2 terminate and then reinitiate at the next downstream site, thus providing a queue of polymerases at the primary initiation site P1. Putative promoter sequences that are AT-rich lie upstream from each site. Termination occurs at two main sites within long pyrimidine sequences downstream from the 23S RNA gene. A processing site for both large RNAs occurs at a secondary structural feature common to other archaebacterial rRNAs. Transcription of 5S RNA starts mainly at P1/5S at the 5' end of the gene with a low level of initiation from two upstream sites; a similar mechanism of initiation-termination-reinitiation appears to operate and the sites are all preceded by AT-rich promoter-like sequences. Termination occurs a few nucleotides beyound the 3' end of the 5S RNA. The mature RNA contains 133 nucleotides, has a 78% GC bp content and exhibits an unusually extended version of helix I with heterogeneous ends. The expression of the rRNA genes shows characteristics of eukaryotes, eubacteria and archaebacteria.

Journal Article↗

PubMed1993

Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro.

The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.

Binding Sites↗