A putative internal promoter in the 16 S/23 S intergenic spacer of the rRNA operon of archaebacteria and eubacteria (original) (raw)
Characterization of intergenic spacers in two rrn operons of Enterococcus hirae ATCC 9790
Journal of bacteriology, 1993
Two DNA restriction enzyme fragments coding for the 3' termini of 16S rRNA, the 5' termini of 23S rRNA, and the intergenic spaces between them in Enterococcus hirae ATCC 9790 were cloned and sequenced. The intergenic space of one of these genes contains a tRNA(Ala) sequence, whereas the other does not. Nevertheless, the intergenic spaces contain several regions that exhibit high levels of sequence homology and are capable of forming structures with similar base pairs. An analysis of Southern blots of chromosomal DNA cut with one and two restriction enzymes indicated that E. hirae has a total of six rrn operons.
Syst Appl Microbiol, 2005
The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5 0-16S-23S-5S-3 0 structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three À10 and À35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.
Applied and Environmental Microbiology, 2005
Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA Glu(UUC) , tRNA Lys(UUU) , tRNA Val(UAC) , and tRNA Ala(GGC) . Five amplicons contained tRNA Glu(UUC) combined with two additional tRNA genes, including tRNA Lys(UUU) , tRNA Val(UAC) , or tRNA Ala(UGC) . Five amplicons contained tRNA Ile(GAU) and tRNA Ala(UGC) . Two amplicons contained tRNA Glu(UUC) and tRNA Ala(UGC) . Two different isoacceptor tRNA Ala genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA Glu(UUC) -tRNA Val(UAC) -tRNA Ala(UGC) and tRNA Glu(UUC) -tRNA Ala(UGC) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.
Regulatory elements downstream of the promoter of an rRNA gene of E. coli
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
Previously we have shown that plasmid constructs carrying a reporter gene fused to the P2 promoter of the E. coli rrnB gene exhibited a strange two-phase kinetics of expression depending on the physiological conditions of the cell if a short DNA region downstream of the promoter was present between the promoter and the reporter gene (Lukacsovich et al. (1987) J. Bacteriol. 169, 272-277). Insertion of a synthetic oligonucleotide corresponding to the first half of this region into constructs where the reporter directly follows the promoter, leads to a complete Mock of expression in vivo, while in vitro -in a purified system -transcription is not inhibited. Band-shift experiments indicate that the putative regulatory region downstream of the promoter specifically binds protein(s) present in total bacterial extracts. 0167-4781/90/$03.50
RNA-polymerase binding at the promoters of the rRNA genes of Escherichia coli
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1980
The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis of polymerase binding, transcription initiation and nitrocellulose filtration of RNA-polymerase-DNA complexes, using restriction endonuclease generated fragments of recombinant plasmids and a transducing phage. The following observations have been made: 1. Two transcription initiation sites have been located approximately 200 and 300 base pairs upstream from the beginning of the sequence coding for mature 16 S rRNA. 2. Polymerase binding at these sites can be observed electronmicroscopically and a 360 base-pair fragment containing these sites binds to nitrocellulose in the presence of RNA-polymerase. This complex dissociates even at moderately high (0.1-0.2 M) salt concentrations. Although transcription initiation is reported to be more frequent at the first of these sites, the binding is much stronger at the second site. 3. In the case of the rrnD gene, BamHI cleaves a few base pairs upstream from the first transcription start site. This cleavage destroys polymerase binding at this site but does not influence binding at the second site. 4. At higher polymerase/DNA ratio four weak but distinct and regularly spaced binding sites can be observed preceding the two initiation sites at approximately 1000, 820, 640 and 440 base pairs before the mature 16 S rRNA sequence. 5. An extremely strong binding site is located about 1300 base pairs upstream from the beginning of the 16 S rRNA sequence. Very little (if any) initiation occurs at this site. The possibility is discussed that the noninitiating binding sites preceding the two transcription start points might functionally belong to the promoter region.
Journal of Molecular Evolution, 1998
The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNAGlu-2 and three (rrnA, D, and H) contain genes for tRNAIle-1 and tRNAAla-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.
Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli
Journal of bacteriology, 1999
The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some ...
1994
The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 165, 235 and 55 rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 165 rRNA and 235 rRNA genes and the spacer-2 region separating the 235 rRNA and 55 rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, 'Mycobacterium lufu ' and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 55 rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase 111 processing sites within the spacer4 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 235 rRNA and for precursor 55 rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 55 rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 165 rRNA sequences in establishing phylogenetic relationships between very closely related species.
rRNA Promoter Activity in the Fast-Growing Bacterium Vibrio natriegens
Journal of Bacteriology, 2002
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 t...
Sequence of the 16 S-23 s spacer region in two ribosomal RNA operons of Escherichia coli
The Journal of biological chemistry, 1979
The transducing phages lambdadaroE and lambdadilv5, which carry the Escherichia coli ribosomal RNA operons rrnD and rrnX, respectively, have been mapped with the restriction endonucleases BamHI, EcoRI, HindIII, and Sma I. Using hybridization techniques, we have located the ribosomal RNA genes on these phage DNAs. The DNA sequence of the 437-base-pair 16 S-23 S ribosomal RNA intergenic spacer in the two rRNA operons rrnD and rrnX has been determined. The nucleotides examined exhibit only one base pair change between rrnD and rrnX. Both spacer regions contain the genes for tRNA1Ile and tRNA1BAla; the gene sequences are identical with the previously deduced tRNA sequences and are clustered within the first 60% of the spacer DNA. The most striking feature of the 16 S-23 S intergenic region in these two operons is the disparity in G-C content between the tRNA gene sequences (60% G-C) and the remaining spacer DNA (37% G-C). Spacer sequences are known to be involved in the processing of th...
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
An archaebacterial promoter sequence assigned by RNA polymerase binding experiments
Canadian Journal of Microbiology, 1989
sequence assigned by RNA Polymerase binding experiments. Can. J. Microbiol. 35: 30-35. To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA Polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA-rRNA genes. The binding region of the RNA Polymerase extends from 30 base pairs (bp) upstream (-30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting transcription factors. The DNA segment protected from nuclease digestion by bound RNA Polymerase contains an octanucleotide sequence centered at -25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA Polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus TTTAjATA as a general element of Promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA Polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro. THOMM, M., WICH, G , BROWN, J. W., FREY, G., SHERF, B. A., et BECKLER, G. S. 1989. An archaebacterial promoter sequence assigned by RNA Polymerase binding experiments. Can. J. Microbiol. 35 : 30-35. Dans le but d'identifier une sequence archaebacterienne de promoteur, des etudes sur la protection contre la nuclease ont ete entreprises avec de TARN Polymerase purifiee de Methanococcus vannielii. Cette enzyme se lie specifiquement ä la fois aux genes qui encodent la proteine (hisA et methyle CoM reductase, composant C) et aux genes ARN t -ARN r . La region d'attachement de TARN Polymerase s'etend depuis 30 bp en amont (-30) ä 20 bp en aval (+20) du site de debut de transcription in vivo. Cette decouverte indique que l'enzyme archaebacterienne reconnait les promoteurs sans Fintervention de facteurs de transcription. Le segment d'ADN protege contre la digestion par la nuclease, grace ä TARN Polymerase liee, contient une sequence de huit nucleotides qui est centree ä -25; cette sequence est conservee entre les genes qui encodent la proteine et les genes d'ARN stable. D'apres l'attachement specifique de Tenzyme aux seuls fragments d'ADN qui contiennent ce motif, nous proposons comme signal de reconnaissance principal de TARN Polymerase de Methanococcus la sequence TTTATATA. Une comparaison de ce motif avec d'autres sequences d'ADN archaebacteriens qui ont fait Pobjet de publications revele la presence de sequences homologues ä la meme localisation en amont de 36 genes. Nous proposons donc 1'Organisation d'ensemble TTTAjATA comme un element commun aux promoteurs chez les archaebacteries. Malgre l'attachement specifique de cette enzyme, la plupart des preparations d'ARN Polymerase de Methanococcus vannielii ne reussisent pas, in vitro, ä amorcer la transcription dans les sites appropries. Nous presentons ici la premiere evidence de l'existence possible d'un facteur de transcription qui confere ä l'enzyme la capacite d'initier et de terminer la transcription, particulierement in vitro. Mots des : promoteur, empreinte, boite de TATA, transcription, ARN Polymerase. [Traduit par la revue]
Journal of Bacteriology, 1989
Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA pro...
Variable rRNA gene copies in extreme halobacteria
Nucleic Acids Research, 1988
Using PFG electrophoresis techniques, we have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence (1). The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.
Journal of Bacteriology
We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnBlp in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.
Nucleic Acids Research, 1988
Transcription initiation of the tisfi sent in aiua. in the archaebacterium MuthinnrnmiT uannlitlii. as determined by nuclease Sj and primer extension analyses, occurs 73 base pairs <bp) upstream of the translation initiation >ita. Binding of U. uannlalii RHA polymerase protects 43 bp of DNA. from 33 bp upstream (-35) to 8 bp downstream <+8) of the hisP mRNA initiation site, from digestion by DNase I and exonuclease III. An fi+T rich region, with a sequence ujhich conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location <-25> upstream of the polgpeptide-encoding bisfi gene. It appears therefore that a TATA-lika sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium.