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At least 829 records · Page 46Linked to original sources
PubMed1989

The conserved 900 stem/loop region in Escherichia coli 16S ribosomal RNA is not required for protein synthesis.

Plasmid pPM114 carries the Escherichia coli 16S ribosomal RNA gene under the control of a T7 promoter. It can generate in vitro transcribed 16S rRNA that can be assembled into functional 30S ribosomal subunits. Two deletion mutants were derived from pPM114, by partial or total deletion of the conserved 900 stem/loop region of the 16S rRNA. These mutants, pMG delta 10 and pMG delta 23, respectively lack bases 895 to 904 and 889 to 911 of the 16S rRNA. The amputated 16S rRNA transcripts synthesized from these mutated plasmids were assembled into 30S subunits which were as active under the direction of an artificial or a natural messenger as subunits reconstructed with the full-length 16S rRNA transcript. They also responded as well to the stimulation of misreading by streptomycin, although the deleted region is proximal to the streptomycin binding domain. However, when we attempted to delete the 895-904 or 889-911 region from the 16S rRNA gene in plasmid pKK3535 which carries the rrnB operon, no transformants harbouring plasmids with one of these deletions could be recovered. These observations suggest that the 900 stem/loop region of the 16S rRNA is not required for the ribosomal function but is probably essential for important cell regulatory functions.

Bacterial Proteins↗
PubMed1987

Organization of the regions flanking the rRNA cistrons of Mycoplasma strain PG50.

Four clones harboring fragments of the rRNA cistrons have been isolated from a genomic library of Mycoplasma PG50 DNA. By heteroduplex and displacement loop analysis, supplemented with sequence data, we demonstrate that the four clones together cover the two rRNA cistrons of Mycoplasma PG50 as well as the flanking regions. Parts of the cloned fragments have been analyzed in detail, and the following conclusions have been reached. Genes for tRNALys and tRNALeu are located about 450 bp upstream of the 16S rRNA gene of rrnA. An open reading frame of at least 215 codons is located just upstream of and in the same direction as rrnB. Analysis of the 3' flanking region of rrnA reveals that no tRNA genes succeed this operon. Also here an open reading frame can be identified, which is at least 83 codons long and has a putative promoter and a possible ribosome binding site. Its direction is the same as that of rrnA. None of the open reading frames has any function assigned yet. The last 880 bp of the 23S and the 5S genes of rrnA are compared with the corresponding Bacillus subtilis sequence and sequences of other Mycoplasma species, respectively.

Base Sequence↗
PubMed1993

A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms.

A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

Base Sequence↗
PubMed1999

The phenotype of mutations of G2655 in the sarcin/ricin domain of 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA forms a part of the site for the association of the elongation factors with the ribosome and hence is critical for the binding of aminoacyl-tRNA and for translocation. The domain is also the site of action of the eponymous toxins which catalyze covalent modification of single nucleotides that inactivate the ribosome. The conformation of the conserved guanosine at position 2655 is an especially prominent feature of the structure of the SRD: the nucleotide is bulged out of a helix and forms a base-triple with A2665 and U2656. G2655 in 23 S rRNA is protected from chemical modification when the elongation factors, EF-Tu and EF-G, are bound to ribosomes and the analog of G2655 in oligoribonucleotides is critical for recognition by the toxin sarcin and by EF-G. The contribution of G2655 to the function of the ribosome has been evaluated by constructing mutations in the nucleotide and determining the phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a deletion of, or transversions in, G2655 is lethal to E. coli cells, whereas a defect in the growth of cells with a G2655A transition is observed only in competition with wild-type cells. The sedimentation profiles of ribosomes with mutations in G2655 are altered; most markedly by deletion or transversion of the nucleotide, less severely by transition to adenosine. Mutations of G2655 confer resistance to sarcin on ribosomes. Ribosomes with G2655Delta, G2655C, or G2655U mutations in 23 S rRNA are not active in protein synthesis, whereas those with the G2655A transition mutation suffer decreased activity.

Base Sequence↗
PubMed1997

Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy.

Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597 x C643 and A596 x U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595 x (A596 x U644). The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598 x A640 base pair and appears to stabilize the G597 x C643 and A596 x U644 base pairs.

Amino Acid Sequence↗
PubMed1989

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli.

We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p 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.

Escherichia coli↗
PubMedDate not supplied

Activity of ribosomal and tRNA promoters of Bacillus subtilis during sporulation.

The rrnB operon in Bacillus subtilis includes 21 tRNA genes downstream of the ribosomal genes. In addition to the dual promoters upstream of these ribosomal genes, a second promoter is found within the tRNA gene region. In this study, these two promoter regions were inserted before the lacZ gene and each was integrated as a single-copy into the B subtilis chromosome to examine their activity during sporulation. Both promoters exhibited similar strength and temporal downregulation from vegetative growth through t3 of sporulation. At t3, transcription from both promoters was approximately 20% of that in logarithmic growth. No obvious function of the second promoter is evident except to boost transcription of downstream genes. This function may be important because the ribosomal promoters of rrnB are weak relative to other ribosomal promoters. For instance, the rrnO promoters were much stronger than the rrnB ribosomal promoters and considerably more active at t3. Thus, all ribosomal promoters are not of the same strength nor are they transcriptionally downregulated to the same extent during sporulation. However, both the rRNA and tRNA promoters in the rrnB operon are similar in strength and downregulation.

Bacillus subtilis↗
PubMed1984

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.

Base Sequence↗
PubMed1993

Origin of 16S and 23S rRNAs and the E. coli str operon, as derived from tandem tRNA repeats.

The bases 1175-1542 (3'-term.) and 94-510 of the E. coli (EC) 16S rRNA are both homologous to the [5S rRNA-tRNA Asn-tRNA(Ser)-tRNA(Glu)-tRNA(Val)-tRNA(Met)] region of the Bacillus subtilis (BSU) trrnD operon, and to the S12 and S7 r-protein-encoding region (S12/S7 region) in the EC str operon. The 2570-2906 (3'-term.) of the EC 23S rRNA is also homologous to the trrnD. These rRNAs had evolved from a str-like pre-mRNA.

Amino Acid Sequence↗
PubMed1997

Sequence and copy number of the Xanthomonas campestris pv. campestris gene encoding 16S rRNA.

A 6.7-kb Sau3A1 fragment containing ribosomal RNA genes was cloned from the chromosome of Xanthomonas campestris pv. campestris strain 17 by a PCR-based strategy. Nucleotide sequence was determined for the 16S rRNA gene (1,544 nt). This gene has a G+C content of 54.9% which is similar to the 16S rRNA genes of Escherichia coli and Pseudomonas aeruginosa but different from the value reported for the whole X. campestris chromosome (64%). Sequence alignment revealed that AGGAGG is consensus for ribosome binding, with the internal GGAG to be paired most frequently with the anti-Shine-Dalgarno sequence. This consensus sequence was found in the regions upstream from the initiation codon of 98 Xanthomonas genes among 116 aligned, but not in the remaining genes. This suggests that about 16% of the Xanthomonas genes do not possess typical ribosome binding sites and another mechanism may be required for recognition of correct translation initiation sites. Two rrn operons were detected in Xc17 chromosome by pulsed-field gel electrophoresis and Southern hybridization.

Base Sequence↗
PubMed2002

Infection with Anaplasma phagocytophila in cervids from Slovenia: evidence of two genotypic lineages.

OBJECTIVE: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined. METHODS: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products. RESULTS: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately. CONCLUSIONS: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.

Anaplasma phagocytophilum↗
PubMed1987

Spectinomycin resistance at site 1192 in 16S ribosomal RNA of E. coli: an analysis of three mutants.

Three different single base substitutions were constructed at residue C-1192 in 16S rRNA on a plasmid-coded rrnB operon of E. coli using site-directed mutagenesis. All 3 mutants conferred different levels of resistance to spectinomycin in transformed cells but none affected the growth rate in the absence of the antibiotic. The G-1192 mutant conferred remarkable resistance, permitting growth in 40 mg/ml of spectinomycin.

Drug Resistance, Microbial↗
PubMed1990

Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.

Base Sequence↗
PubMed1997

Targets for pSAM2 integrase-mediated site-specific integration in the Mycobacterium smegmatis chromosome.

An improved integrative cassette from plasmid pSAM2 has been constructed containing plasmid int and attP genes but excluding the xis gene, which should results in increased stability by suppression of the excision reaction. This cassette was included in both suicide and thermosensitive plasmids and used for integration in Mycobacterium smegmatis. Suicide plasmids containing this cassette integrated at a single site (attB1) in the M. smegmatis chromosome. The sequence of the attB1 site has been determined and was identified as a putative tRNA(Pro) gene. Thermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to simultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.

Base Sequence↗
PubMed2003

Dispersal and phylogenetic diversity of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA-intergenic spacer sequence analyses.

More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.

Bacterial Proteins↗
PubMed1988

Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli.

The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

Escherichia coli↗
PubMed2006

Mapping contacts of the S12-S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli.

In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the 'streptomycin' operon transcript by binding to the S12-S7 intercistronic region. Here, we describe the contacts of N-terminally His(6)-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(-34), U(-35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12-S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding.

Base Sequence↗