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At least 1,171 records · Page 65Linked to original sources
PubMed2003

RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.

5' Untranslated Regions↗
PubMed1989

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli.

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

Ampicillin↗
PubMed1996

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.

Base Sequence↗
PubMed2006

Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics.

Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected.

Anti-Bacterial Agents↗
PubMed1995

Genetic basis of macrolide resistance in Mycobacterium avium isolated from patients with disseminated disease.

Clarithromycin (CLM) and azithromycin (AZM) are important agents in the treatment of disseminated Mycobacterium avium complex disease; however, monotherapy with these macrolides often leads to clinically significant resistance. The underlying resistance mechanism was investigated by comparing 23S rRNA gene sequences in the domain V region of 10 CLM-susceptible strains included in this study. The only differences in the domain V sequences associated with CLM resistance were at position 2274 of the complete M. avium 23S rRNA gene (GenBank accession no. X74494). All the CLM-susceptible strains had an A residue at this site, whereas seven of the eight CLM-resistant strains had either a C, G, or T. Four of these seven CLM-resistant strains emerged during monotherapy with CLM and two emerged during AZM monotherapy, showing that resistance selected by either macrolide was associated with mutation of the 23S rRNA gene. Thermodynamic analysis of secondary rRNA structure suggests that the observed mutations cause an alteration in free energy associated with rRNA folding, which may result in a localized conformation change in assembled ribosomes. Such a shift may be important in the resistance of ribosomes to the effects of macrolides. This study therefore establishes a link between mutations within the 23S rRNA gene and clinically significant macrolide resistance in M. avium and also identifies a possible molecular mechanism of resistance at the level of the ribosome.

AIDS-Related Opportunistic Infections↗
PubMed1982

Autogenous control: ribosomal protein L10-L12 complex binds to the leader sequence of its mRNA.

Ribosomal proteins L10 and L12 are encoded in the L10 operon, situated at position 89.5 min on the Escherichia coli genetic map, and are able to regulate their own translation. The two proteins form a L10-L12 complex that is able to bind specifically to the leader sequence of the L10 operon mRNA and prevent translation. We show that the leader sequence: (i) is required for the translation of mRNA into L10 and L12 proteins; and (ii) contains a unique binding site for the inhibitory L10-L12 complex. We suggest that a specific secondary structure of the leader RNA is required for translation. When this structure is perturbed by L10-L12 binding, by deletion, or point mutations, translation is inhibited. The block on the synthesis of L10 and L12 can presumably be removed by the incorporation of the inhibitory L10-L12 complex into assembling 50S ribosome subunits. We observed that rRNA prevents the binding of L10-L12 to the mRNA. Furthermore, we have identified extended sequence homologies within the 23S rRNA and L10 leader region RNA. The L10-L12 binding site on the mRNA includes part of the homologous sequences.

Bacterial Proteins↗
PubMed2003

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes.

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.

Amino Acid Sequence↗
PubMed1994

Novel growth rate control of dam gene expression in Escherichia coli.

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested. In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation. The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.

Base Sequence↗
PubMedDate not supplied

Molecular basis of ribotype variation in the seventh pandemic clone and its O139 variant of Vibrio cholerae.

Ribotyping has been widely used to characterise the seventh pandemic clone including South American and O139 variants which appeared in 1991 and 1992 respectively. To reveal the molecular basis of ribotype variation we analysed the rrn operons and their flanking regions. All but one variation detected by BglI, the most discriminatory enzyme, was found to be due to changes within the rrn operons, resulting from recombination between operons. The recombinants are detected because of the presence of a BglI site in the 16S gene in three of the nine rrn operons and/or changes of intergenic spacer types of which four variants were identified. As the frequency of rrn recombination is high, ribotyping becomes a less useful tool for evolutionary studies and long term monitoring of the pathogenic clones of Vibrio cholerae as variation could undergo precise reversion by the same recombination event.

Bacterial Typing Techniques↗
PubMed1984

Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences.

We have localized the antitermination system involved in E. coli ribosomal RNA transcription and compared it with antitermination in the lamboid bacteriophages. In vivo experiments with gene-fusion plasmids were used to examine the ability of specific areas of the rrnG control region to convert an ordinary transcription complex into antitermination transcription complex. A 67 bp restriction fragment immediately following the rrnG P2 promoter decreased transcription termination about 50%. This fragment contains box A-, box B-, and box C-like sequences similar to those in lambda nut loci. It also caused transcripts from lac and hybrid trp-lac promoters to read through a transcription terminator. Translation through the 67 bp segment or reversal of its orientation resulted in complete loss of antitermination activity. We conclude that the E. coli ribosomal RNA operons possess an antitermination system similar to that used by the bacteriophage lambda.

Bacteriophage lambda↗
PubMedDate not supplied

[Rearrangement of nucleoli in hepatocytes stimulated for proliferation and enhancement of their transcription function].

The nucleoli of normally functioning guinea-pig hepatocytes that have a nucleolonemal (strand-like) organization differ from identical nucleoli of other cells. Their nucleolonema consists as a rule of a fibrillar component with 45S RNA and is poor in granulas that contain pre-rNA molecules of an intermediate size and 28S rRNA, a dense fibrillar component with nascent rRNPs in its composition was not revealed. In hepatocytes stimulated by a 2/3 liver resection rearrangements in nucleoli were found. This brought to a conclusion that rRNA metabolism undergoes some changes. In 2.5 and 5 hours after the resection the hepatocytes' nucleoli were characteristic of a greater thickness of strands and a smaller size of vacuoles, appearance of distinct zones of the dense fibrillar component and an increased amount of RNP-granules. All these observations taken together point out at an increased synthesis and processing of rRNA at early stages of the prereplicative period. In 9 hours the character of changes in nucleoli was different: the vacuoles were considerably widened, whereas the thickness of strands that consisted of a well-expressed dense fibrillar, fibrillar and granular components was lesser. Such rearrangement points out at an increased transport of preribosomes from the nucleolus, a high level of synthesis and processing of nascent RNP-product being maintained. The changes of nucleolar RNP-component were followed by appearance of greater blocks of perinucleolar condensed chromatin, which may be connected with "cutting-off" some tissue-specific genes and initiation of functioning of the mitotic operon genes.

Animals↗
PubMed2002

The dawn of gene isolation.

Ever since it became clear through the work of Watson and Crick that the gene is a stretch of double stranded helical DNA and is understandable in chemical terms, biochemists have striven to get their hands on isolated genes. The isolation of the ribosomal genes of Xenopus laevis in 1966 provided a first instance where a purified DNA of known function could be investigated, long before the advent of gene cloning technologies. The second instance was the purification of the Lac operon from Escherichia coli. Later, but still before the gene cloning days the 5S RNA genes of X. laevis and the histone genes of the sea urchin Psammechinus miliaris were isolated by physico-chemical methods, but their isolation marked the end of an era. By 1975, gene cloning technology was well established and the isolation of genes quickly became an everyday occurrence.

Animals↗
PubMed1986

In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast.

The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.

Base Sequence↗
PubMed1982

Transcription of cloned Xenopus laevis ribosomal DNA microinjected into Xenopus oocytes, and the identification of an RNA polymerase I promoter.

Transcription of a cloned Xenopus laevis ribosomal DNA (rDNA) fragment, microinjected into Xenopus oocytes, is initiated at the in vivo 40S pre-ribosomal RNA (pre-rRNA) site (+/- 2 bp) by RNA polymerase I. An X. laevis RNA polymerase I promoter has been mapped by studying the transcription of in vitro rDNA mutants in the oocyte system. The active promoter lies within the DNA segment beginning 145 bp upstream, and most probably ending 16 bp downstream, from the 40S pre-rRNA initiation site (-145 bp to +16 bp). Furthermore, active promoter elements lie more than 35 bp upstream from the initiation site (-35 bp). The X. laevis RNA polymerase I promoter therefore lies mainly upstream from the 40S pre-rRNA initiation site. Independent deletion of three adjacent rDNA segments lying between -61 and +16 bp reduces promoter activity by a factor of more than 16. The central of these "null" deletions removes an oligo(T)6 motif at -27 bp that is in an analogous position to the Goldberg-Hogness (TATA) box of RNA polymerase II promoters.

Animals↗
PubMed1990

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm.

The nucleotide sequence and the 5' flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria, the P. littoralis rbcL gene is more closely related to that of a beta-purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996-4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599-608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of the rrnG ribosomal RNA promoter region of Escherichia coli.

The primary structure of the promoter region for a ribosomal RNA transcription unit (rrnG) of Escherichia coli K12 has been determined. The sequence was obtained from 1 1.5 kbp EcoRI fragment derived from the hybrid plasmid pLC23-30. This fragment contains 455 bp preceding P1 of the rrnG promoter region and 674 bp of the rrnG 16S RNA gene. The sequence before the rrnG promoter region contains an open reading frame (ORF-BG) followed by a possible hairpin structure that resembles other known transcription terminators. The sequence of the rrnG promoter region is similar but not identical to that of rrnA and rrnB. Several minor differences between the sequences of the 16S RNA genes of rrnG and rrnB were also noted. In addition, sequences were found that could generate special structures involving the promoter regions of rrn loci. Such structures are described and their possible involvement in the regulation of ribosomal RNA synthesis is discussed.

Base Sequence↗
PubMed1994

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Codon↗
PubMed1999

Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8. Generation of a mutant lacking ribosomal protein S17.

S17 is a primary rRNA-binding protein which has been implicated in ribosome assembly and translational fidelity. We describe the generation and biochemical characterization of an S17 minus ribosomal mutant, a ribosomal protein-lacking mutant obtained in Thermus thermophilus HB8. The S17 mutant was obtained by insertional inactivation of the target gene with the kanamycin adenyl transferase (kat) gene, making use of a Thermus-Escherichia shuttle vector and the natural ability of Thermus to transform. In the final construct used to transform Thermus cells, the S17 coding region was replaced with the kat gene cloned in-frame with the first three amino acids of S17. Hence, in vivo transcription of the kat gene was under the control of the ribosomal operon promoter. As in Escherichia coli, the Thermus S17 mutant exhibited a temperature-sensitive phenotype. Two-dimensional PAGE, Western blot, and ELISA confirmed the absence of S17 from the mutant ribosomes. Sucrose-gradient profiles of mutant cells showed a clear separation and normal proportions of 50S and 30S subunits and a normal ratio between them. In addition, the S17 mutant showed the presence of a 20S peak representing assembly-defective particles. The successful re-incorporation of protein S17 into the mutant ribosomes was demonstrated when reconstitution with isolated S17 was performed at 60 degrees C.

Anti-Bacterial Agents↗