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At least 595 records · Page 33Linked to original sources
PubMed1995

The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter.

The E. coli rrnB P1 promoter owes its strength, in part, to the transcriptional activator protein FIS. FIS binds to three sites upstream of the RNA polymerase (RNAP) binding site and increases transcription in vivo four to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnB P1. We also find that FIS does not bind cooperatively to its three sites upstream of rrnB P1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I.

Base Sequence↗
PubMed1997

Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA.

RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome profiles upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our findings contradict previously reported properties of individual mutants. Particularly notable is our finding that the first reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing deltaC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.

Base Sequence↗
PubMed1997

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Amino Acid Sequence↗
PubMed1999

Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors.

The sarcin/ricin domain in 23 S/28 S rRNA is crucial for ribosome function, since it constitutes at least part of the binding site for the elongation factors and hence is essential for binding aminoacyl-tRNA and for translocation. The domain is also the site of action of ricin and sarcin and analysis of the effect of mutations in the RNA on recognition by the cytotoxins has helped to define the structure and to understand the function of the region. We have constructed deletions, separately, of pairs of non-conserved, juxtaposed but non-hydrogen-bonded nucleotides that correspond to C4317 and C4331, and to U4316 and C4332, in an oligoribonucleotide that mimics the sarcin/ricin domain in rat 28 S rRNA. The deletions had no effect on the depurination of A4324 by ricin nor on the cleavage of the phosphodiester bond on the 3' side of G4325 by sarcin. However, simultaneous deletion of the four nucleotides decreased cleavage by sarcin but did not affect depurination by ricin. Removal of the non-canonical A4318.A4330 pair abolished recognition by both toxins. Deletion from oligoribonucleotides, that reproduce the sarcin/ricin domain of Escherichia coli 23 S rRNA, of U2653 and C2667 (equivalent to U4316, C4317 and C4331, C4332 in 28 S rRNA), or substitution of guanosine for U2653 (designed to form a Watson-Crick G2653.C2667 pair), reduced cleavage by sarcin whereas depurination by ricin was slightly increased. An increase in the stability of the mutant oligoribonucleotides may be the basis of the impairment in sarcin action. The tm for the wild-type RNA is 60 degreesC; for the double-deletion mutant U2653Delta/C2667Delta it is 65 degreesC; and for the U2653G transversion it is 69 degreesC. Expression of a mutant 23 S rRNA gene lacking U2653 and C2667 is lethal and a U2653G transversion mutation impairs growth. The mutant ribosomes are less active in protein synthesis than the wild-type and ribosomes with the U2653G mutation are resistant to sarcin. The binding of EF-G to oligoribonucleotides with a U2653/C2667 double deletion is reduced and an effect on the affinity of the factor for the sarcin/ricin domain may account in part for the decrease in ribosome efficiency. The results stress the potential importance in rRNA structure and function of non-conserved nucleotides, and suggest that the sarcin/ricin domain in ribosomes requires a region of structural flexibility for optimal efficiency.

Animals↗
PubMed2000

The bacterial DNA-binding protein H-NS represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex.

The interaction of the bacterial regulatory protein H-NS with RNA polymerase and the ribosomal RNA P1 promoter was analyzed to better understand the mechanism of H-NS-dependent transcriptional repression. We could show that initial binding of RNA polymerase to the promoter was not inhibited by the simultaneous interaction of H-NS, although H-NS binding sites extend into the core promoter region. Binding of sigma(70)-saturated RNA polymerase and H-NS to the promoter DNA occurs cooperatively and results in a stable complex of slower gel electrophoretic mobility as compared to complexes formed with the single proteins. The presence of the upstream curved H-NS binding site contributes strongly to the cooperative RNA polymerase-promoter interaction. By KMnO(4) modification of single-stranded template nucleotides we could show that open complex formation at the rrnB P1 promoter was not inhibited by H-NS binding. An increased KMnO(4) reactivity of several positions within the open complex rather supports the view that open complex formation is stimulated in presence of H-NS. Moreover, subtle changes in the modification pattern indicate that the open complex formed in the presence of H-NS are structurally distinct from the H-NS-free complex. In vitro transcriptional analysis of the abortive and productive yields revealed that the formation of transcription products longer than three nucleotides is dramatically reduced in the presence of H-NS, while the amount of shorter abortive products remained unaffected. Together the results demonstrate that H-NS inhibits transcription at the rrnB P1 promoter not by interfering with initial RNA polymerase binding but by blocking chain elongation steps subsequent to the first (two) phosphodiester bond formations. The mechanism of H-NS dependent repression at rRNA promoters can thus be explained as a trap which inhibits substrate NTP incorporation beyond template position +3 into the initial transcribing complex.

Allosteric Site↗
PubMed2002

Architecture of Fis-activated transcription complexes at the Escherichia coli rrnB P1 and rrnE P1 promoters.

The transcription factor Fis activates the Escherichia coli rRNA promoters rrnB P1 and rrnE P1 by binding to sites centered at -71 and -72, respectively, and interacting with the C-terminal domain of the alpha subunit of RNA polymerase (RNAP alphaCTD). To understand the mechanism of activation by Fis at these promoters, we used oriented alpha-heterodimeric RNAPs and heterodimers of Fis to determine whether one or both subunits of alpha and Fis participate in the alphaCTD-Fis interaction. Our results imply that only one alphaCTD in the alpha dimer and only one activation-proficient subunit in the Fis dimer are required for activation by Fis. A library of alanine substitutions in alpha was used to identify the alphaCTD determinants required for Fis-dependent transcription at rrnB P1 and rrnE P1. We propose that the transcriptional activation region of the promoter-proximal subunit of the Fis dimer interacts with a determinant that includes E273 of one alphaCTD to activate transcription. We further suggest that the Fis contact to alphaCTD results in alphaCTD interactions with DNA that differ somewhat from those that occur at UP elements in the absence of Fis. The accompanying paper shows that the 273 determinant on alphaCTD is also targeted by Fis at the proP P2 promoter where the activator binds overlapping the -35 hexamer. Thus, similar Fis-alphaCTD interactions are used for activation of transcription when the activator is bound at very different positions on the DNA.

Base Sequence↗
PubMed1998

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum.

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.

Animals↗
PubMed1993

Zymobacter palmae gen. nov., sp. nov., a new ethanol-fermenting peritrichous bacterium isolated from palm sap.

Zymobacter palmae gen. nov., sp. nov. was proposed for a new ethanol-fermenting bacterium that was isolated from palm sap in Okinawa Prefecture, Japan. The bacterium is gram-negative, facultatively anaerobic, catalase-positive, oxidase-negative, nonsporeforming and peritrichously flagellated. It requires nicotinic acid for growth. It ferments hexoses, alpha-linked di- and tri-saccharides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannitol and sorbitol). Fifteen percent of maltose in broth medium is effectively fermented, whereas glucose with a concentration higher than 10% delayed growth initiation and decreased growth rates. Maltose is fermented to produce ethanol and CO2 with a trace amount of acids. Approximately 2 mol of ethanol are produced from 1 mol moiety of hexose of maltose. The organism possesses ubiquinone-9. The G + C content of the DNA is 55.8 +/- 0.4 mol%. Major cellular fatty acids were palmitic and oleic acids and cyclopropanic acid of C19:0. Characteristic hydroxylated acid was 3-hydroxy dodecanoic acid. The bacterium is distinct from other ethanol-fermenting bacteria belonging to the genera Zymomonas Kluyver and van Niel 1936 and Saccharobacter Yaping et al. 1990 with respect to chemotaxonomic and other phenotypic characters to warrant to compose a new genus and a new species. The type strain is strain T109 (= IAM 14233).

Base Sequence↗
PubMed1993

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria.

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character "intracytoplasmic membrane structure", that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.

Base Sequence↗
PubMed1992

Detection of variation in the ribosomal RNA gene clusters by a modified fluorescence in situ hybridization method.

Physical mapping of genes by fluorescence in situ hybridization (FISH) has become routine using fluorescein isothiocyanate (FITC) for probe detection and propidium iodide (PI) for chromosome staining. We have modified this conventional FISH method in a way that utilizes Texas red (TR) for signal detection and quinacrine mustard (QM) for chromosome banding. Using this Texas red and quinacrine (TRQ) method, we were able to identify individual acrocentric chromosomes with varying degrees of ribosomal RNA gene clusters. Two acrocentric chromosomes were found to carry extremely small number of rRNA gene copies as compared to the other eight counterparts in human diploid lymphoblastoid cell line GM00130B. Thus, the TRQ method allows one to probe for a specific sequence while identifying individual chromosomes and will be powerful for the chromosomal localization of various genes.

Cell Line↗
PubMed1998

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus.

Multiple introns were detected in the 16S rRNA gene of newly isolated Thermoproteus species strains IC-033 and IC-061 and Thermoproteus neutrophilus JCM 9278. In the 16S rRNA gene of strain IC-033, five introns of 627, 762, 636, 33, and 682 bp existed after positions 548, 781, 1092, 1205, and 1213 (according to the Escherichia coli numbering system), respectively. Likewise, strain IC-061 possessed 764-, 32-, and 688-bp introns after positions 781, 1205, and 1213, respectively; and T. neutrophilus JCM 9278 had 34- and 663-bp introns after positions 1205 and 1213, respectively. All the introns carried the putative intron core structures consisting of a bulge-helix-bulge motif and a long stable stem. The large introns carried open reading frames containing the LAGLI-DADG-like motifs in their terminal inserts; however, three out of four large introns of strain IC-033 seemed to incur frameshift mutations. Occurrence of introns at the same insertion sites in the three strains would allow tracing of the evolutionary movements of these introns.

Evolution, Molecular↗
PubMed1996

Functional promoter elements common to ribosomal protein and ribosomal RNA genes in Neurospora crassa.

The promoter sequences of a cytoplasmic ribosomal protein gene (crp-2) of Neurospora crassa were identified using promoter deletion and substitution mutants. A gene-targeting strategy was used to assay the mutants in vivo. The promoter architecture of crp-2 is complex and is more similar to that of ribosomal protein genes in mouse than in Saccharomyces cerevisiae. Six regions were identified as important for transcription. These included two elements, a CG repeat and a Dde box, that are conserved in most other promoters of N. crassa ribosomal protein genes and have also been demonstrated as being required for transcription from the 40 S rRNA promoter by RNA polymerase I in vitro. The CG repeats located at -73 to -66 and between -189 and -154 were functionally redundant and increased transcription efficiency by 10- to 15- fold. The Dde boxes located at -153 to -147 and at -95 to -83 contributed 2-fold and 5-fold to transcription efficiency, respectively. An unidentified element between -254 and -190 contributed 2-fold, while a pyrimidine-rich region between -85 and -66 influenced the start point of transcription.

Base Sequence↗
PubMed1995

Evaluation of the E. coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study.

A concept study devised for the development of a biological containment system has been conducted. We show that the lysis genes of different phage origin function in a variety of bacteria. They may therefore be suited for conditional suicide cassettes. Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E. coli production strains from a bioreactor. Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off. These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions. A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.

Amino Acid Sequence↗
PubMed1991

Structure and organization of ribosomal DNA.

Three trends are seen in the organization of ribosomal DNA genes during evolution: 1) gradual separation and separability of the regulation of transcription of 5S and larger subunit rRNAs; 2) retention of a transcription unit containing both large and small rRNAs; and 3) clustering of genes for both 5S and 18S-28S rDNAs, with the possible association of other 'non-rDNA' in the clusters of 18S-28S rDNA genes by the time mammals evolve.

Animals↗
PubMed1994

Functional analysis of transcription of the Mycobacterium tuberculosis 16S rDNA-encoding gene.

A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study. RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth. Transcription start points (tsp) were mapped by primer extension analysis of RNA from M. tuberculosis H37Rv and M. tuberculosis H37Ra. An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M. tuberculosis H37Rv and H37Ra. One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter. The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E. coli. The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene (cat) in a shuttle plasmid vector, pSD7. The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M. smegmatis. The M. smegmatis transformant exhibited a CAT activity of 16,669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength. Two tsp utilized in M. tuberculosis were also employed in M. smegmatis. The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.

Base Sequence↗
PubMed2006

rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase.

Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that sigma subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the -10 element (within the "discriminator" region). By making a nonoptimal sigma1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.

Base Sequence↗
PubMed2006

The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding.

LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP-rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP-nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP-DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes.

Allosteric Regulation↗