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PubMed1989

Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris.

The ribosomal RNA operons (rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA(Ile), tRNA(Ala),23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3'-ends of the tRNAs are added.

Animals↗

PubMed1982

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.

DNA, Recombinant↗

PubMed1997

Detection of heterogeneity of 18S rRNA inter-genes and mutation arising during PCR amplification.

Direct sequencing revealed sequence heterogeneity among ribosomal RNA gene (rDNA) operons, consisting of 8 base heterogeneous sites on the 18S rDNA of Galactomyces citri-aurantii IFO 10822, and 6 base heterogeneous sites in the same region on the 18S rDNA of G. citri-aurantii IFO 10821. Sequence analysis of the cloned 18S rRNA genes of 14 species (19 strains) of ascomycetous yeast-like fungi detected a total of 32 substitutions between two cloned sequences from each of 10 strains. Eight substitutions came from heterogeneity of G. citri-aurantii IFO 10822, and 24 substitutions were predicted to be due to misincorporation by the Taq DNA polymerase. A low frequency of random substitution, estimated to occur in PCR at approximately 1 in 2690 nucleotides, was detected; and transitions occurred 7 times more frequently than transversions.

DNA, Ribosomal↗

PubMed2002

Comparative anatomy of a regulatory ribosomal protein.

Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.

Amino Acid Sequence↗

PubMed2001

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

5' Untranslated Regions↗

PubMed2006

Polyamine Modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines.

We have recently proposed an idea to explain how polyamines enhance cell growth in Escherichia coli. Since most polyamines exist as polyamine-RNA complexes, our idea is that polyamines stimulate several kinds of protein synthesis which are important for cell growth at the level of translation. We found that synthesis of oligopeptide binding protein (OppA), which is important for nutrient supply, adenylate cyclase (Cya), RNA polymerase sigma(38) subunit (RpoS), transcription factor of iron transport operon (FecI), and transcription factor of growth-related genes including rRNA and some kinds of tRNA synthesis (Fis) was enhanced by polyamines at the level of translation. We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were up-regulated by polyamines. Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis. This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli.

Adenylyl Cyclases↗

PubMed2001

Diversity of toxic and nontoxic nodularia isolates (cyanobacteria) and filaments from the Baltic Sea.

Cyanobacteria of the genus Nodularia form toxic blooms in brackish waters worldwide. In addition, Nodularia spp. are found in benthic, periphytic, and soil habitats. The majority of the planktic isolates produce a pentapeptide hepatotoxin nodularin. We examined the morphologic, toxicologic, and molecular characters of 18 nodularin-producing and nontoxic Nodularia strains to find appropriate markers for distinguishing the toxic strains from the nontoxic ones in field samples. After classical taxonomy, the examined strains were identified as Nodularia sp., Nodularia spumigena, N. baltica, N. harveyana, and N. sphaerocarpa. Morphologic characters were ambiguous in terms of distinguishing between the toxic and the nontoxic strains. DNA sequences from the short 16S-23S rRNA internally transcribed spacer (ITS1-S) and from the phycocyanin operon intergenic spacer and its flanking regions (PC-IGS) were different for the toxic and the nontoxic strains. Phylogenetic analysis of the ITS1-S and PC-IGS sequences from strains identified as N. spumigena, and N. baltica, and N. litorea indicated that the division of the planktic Nodularia into the three species is not supported by the ITS1-S and PC-IGS sequences. However, the ITS1-S and PC-IGS sequences supported the separation of strains designated N. harveyana and N. sphaerocarpa from one another and the planktic strains. HaeIII digestion of PCR amplified PC-IGS regions of all examined 186 Nodularia filaments collected from the Baltic Sea produced a digestion pattern similar to that found in toxic isolates. Our results suggest that only one planktic Nodularia species is present in the Baltic Sea plankton and that it is nodularin producing.

Bacterial Toxins↗

PubMed1992

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.

Base Sequence↗

PubMed2005

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.

Bacterial Proteins↗

PubMed1991

Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae.

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

Amitrole↗

PubMed1993

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.

Bacillus subtilis↗

PubMedDate not supplied

Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA.

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3' domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within the 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 degrees C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

Codon, Nonsense↗

PubMed1998

Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of Streptomyces griseus.

A 5018-bp DNA fragment of the rpl/rpo BC gene cluster (here called the rif cluster) of Streptomyces griseus N2-3-11 was analysed by DNA sequencing and transcription studies. By sequence comparison of the deduced proteins, five genes and part of an open reading frame (orf) were identified. The genes encoding the ribosomal (r-) proteins L1 (rplA), L7/12 (rplJ), L10 (rplK) and L11 (rplL), a protein of known function (orf31), and the N-terminus of the beta subunit of RNA polymerase (rpoB), are organized in three operons, rplKA, rplJL and rpoB(C), and the monocistronic transcription unit orf31. The promoters of these transcription units, rplKp, orf31p, rplJp, and rpoBp, were identified and the growth-phase dependence of the transcription of these operons was analysed. Binding sites for the ribosomal proteins L1 and L10 were identified by sequence comparison, suggesting that the r-proteins RplA and RplJ are involved in feedback regulation of their respective operons by binding to specific RNA-binding sites present in both the mRNA and the 23S rRNA, as has been described for other bacteria. The analyses of the rpoBp promoter by means of promoter-probe plasmids suggested a possible attenuator-based regulatory mechanism for the transcription of the rpoB(C) operon.

Amino Acid Sequence↗

PubMed2002

Pulsed-field gel electrophoretic analysis of the genome of Lactobacillus gasseri ATCC33323, and construction of a physical map.

Some species of Lactobacillus are of major industrial and health significance as fermenting agents in the manufacturing of food products, as food preservatives, as "probiotic" bacteria or as vaccine delivery vehicles. In spite of their importance, there is a paucity of published information on their genome organization and structure. In this study, a combination of pulsed field gel electrophoresis (PFGE) and hybridization approaches was used to investigate the genome of L. gasseri neotype strain ATCC33323. PFGE analysis of chromosomal DNA (after digestion with the rare-cutting restriction enzymes I-CeuI, CspI, SmaI, ApaI, and SgrAI) allowed the chromosome size of L. gasseri to be estimated at 1.96 Mbp, and also revealed the presence of a linear plasmid of 48.5 kbp. A physical map of the L. gasseri chromosome, containing 6 sites for the enzymes I-CeuI and 12 for CspI, was constructed. Placed on the map were the genes dnaA and gyrB (usually located close to the origin of replication on the bacterial chromosome) and 18 ribosomal RNA (rrn) genes. Mapping analysis also revealed that the chromosome contained six rrn operons, and that one of them was inverted in orientation with respect to the others. Each rrn operon contained a single copy of each of the three rrn genes, 23S rRNA (rrl), 16S rRNA (rrs) and 55 rRNA (rrf) gene. The constructed physical map should be a useful foundation for genomic and genetic studies of the lactobacilli and provides a platform for applied research, such as the engineering of Lactobacillus strains with improved characteristics for industrial and probiotic applications.

DNA Restriction Enzymes↗

PubMed1996

Ribosomal protein L4 from Escherichia coli utilizes nonidentical determinants for its structural and regulatory functions.

Escherichia coli ribosomal protein L4 has two functions: it is a structural component of the 50S ribosomal sub-unit and it is a repressor of both transcription and translation of its own transcription unit, the 11-gene S10 operon. Genetic and biochemical studies have suggested that L4 can interact with 23S rRNA as well as with both RNA interactions. However, no significant similarities between its two RNA targets can be found at the primary or secondary structure level. To test if identical determinants of L4 are involved in both ribosome assembly and autogenous control, we have isolated L4 mutants defective in either of these functions and asked if a mutant protein divested of one function is also deficient in the other. Several mutations eliminated autogenous control, but still allowed assembly of the mutant L4 protein into functional ribosomes. Conversely, several mutant L4 proteins that could not be detected in 50S subunits nevertheless could regulate expression of the S10 operon. These results indicate that the L4 determinants required for autogenous regulation and ribosome incorporation are not congruent.

Amino Acid Sequence↗

PubMed2003

Ecosystem-dependent adaptive radiations of picocyanobacteria inferred from 16S rRNA and ITS-1 sequence analysis.

Small, coccoid and rod-shaped Synechococcus-type cyanobacteria with either phycoerythrin or phycocyanin as major accessory pigments were isolated from several large, temperate-zone lakes and the brackish Baltic Sea. The picocyanobacteria had two ribosomal operons with a long internal transcribed spacer (ITS-1) separating the 16S rDNA and 23S rDNA. A 16S rRNA-based phylogenetic analysis assigned all isolates to the picophytoplankton clade [sensu Urbach, E., Scanlan, D. J., Distel, D. L., Waterbury, J. B. & Chisholm, S. W. (1998). J Mol Evol 46, 188-201], which also comprises marine Synechococcus spp. and Prochlorococcus spp. The strains assorted to five paraphyletic clusters each containing two or more strains with 99.4-100 % 16S rRNA sequence identity. Five corresponding strain clusters were deduced from analysis of ITS-1 sequences. Sequence divergence in ITS-1 varied between 23 % in the most divergent and 1 % in the phylogenetically most conserved cluster. Clustered strains with low sequence divergence in ITS-1 were frequently isolated from a single ecosystem or hydrographically comparable lakes in the same region. They represent physiologically distinct ecotypes of species which, among other phenotypic variations, frequently differed in their major accessory pigments, the phycobiliproteins. The reproduction of the various pigment traits in different lineages was not correlated with the phylogenetic divergence deduced from 16S rRNA or ITS-1 sequences but rather seemed to be related to characteristics of the ecosystem and habitat from which the strains were isolated. The occurrence of a comparable spectrum of phenotypes in different lineages and ecosystems indicates that different strain clusters developed similar ecotypes during independent adaptive radiations.

Adaptation, Physiological↗

PubMed1985

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro.

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.

Autoradiography↗

PubMed2006

The complete chloroplast genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins of chlorarachniophyte and euglenid secondary endosymbionts.

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.

Animals↗