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At least 325 records · Page 18Linked to original sources
PubMed1987

Purified RNA polymerase II recognizes specific termination sites during transcription in vitro.

We have studied the ability of certain well-defined prokaryotic DNA sequences to act as specific termination signals for highly purified calf thymus RNA polymerase II. We used duplex DNA fragments modified to direct efficient and specific transcription of defined DNA templates to follow transcription with RNA polymerase alone in the absence of additional protein factors. Elongation of RNA chains by RNA polymerase II is processive through most DNA sequences. However, certain DNA sequences serve as effective "intrinsic" terminators for RNA polymerase II; in this they resemble the "rho-independent" terminators for the bacterial RNA polymerase. Several rho-independent bacterial terminators are also able to act as termination signals for RNA polymerase II. However, there is no apparent correlation between the efficiency of termination for the bacterial enzyme and that found for the calf thymus enzyme. One very efficient bacterial terminator (phage T7 early terminator) gives no termination with RNA polymerase II, and we have identified at least two sites that cause the eukaryotic enzyme to terminate but have no effect on transcription by the bacterial enzyme. Hence, the signals recognized as intrinsic termination sites for the two enzymes are substantially different. All of the sites that act as intrinsic terminators for RNA polymerase II contain a series of consecutive thymidine residues in the nontranscribed DNA strand (T-run), and the 3' end of the completed RNA normally lies within this sequence. It is plausible that the T-run is part of the signal for an RNA polymerase II termination site; however, there is no apparent correlation between the number of T residues and the efficiency of the terminator, suggesting that other sequence elements are required for, or modulate, termination. Several lines of evidence suggest that the formation of RNA secondary structures in the nascent transcript is not an essential element of the intrinsic RNA polymerase II termination signal.

Animals↗
PubMed1965

LOCALIZATION OF SPORE MARKERS ON THE CHROMOSOME OF BACILLUS SUBTILIS.

Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Localization of spore markers on the chromosome of Bacillus subtilis. J. Bacteriol. 89:1065-1067. 1965.-Attempts were made to locate spore markers on the chromosome by determining the mapping distance of ery(r), str(r), phe, tyr, and ser markers to which the spore markers are closely linked. The results obtained indicated that locus sp N2-2 is located near the terminus and loci sp 170-2, sp H12-4, and sp 1 are distributed between the middle and the origin of the chromosome. These spore markers are not linked to each other. The above observation confirms our previous finding that in Bacillus subtilis spore markers are distributed randomly along the chromosome.

Bacillus subtilis↗
PubMed2002

Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13.

AIMS: To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13. METHODS AND RESULTS: RNA and DNA of Escherichia coli 536 cells were extracted and fluorimetrically quantified to compare the different contents, throughout a 24 h culture, with their SYTO 13 fluorescence emission when analysed by the cytometer. SYTO 13 fluorescence varied depending on the stage of bacterial growth and in accordance with both DNA and RNA content. RNA content accounted for at least two-thirds of the total fluorescence of a cell. Escherichia coli cells were treated with chloramphenicol to improve their RNA content. With this treatment, both nucleic acids remained constant but there was a clear improvement in fluorescent emission. SYTO 13 fluorescence was also studied in E. coli X-1488 minicells. CONCLUSIONS: Although both nucleic acids are implicated, RNA accounts for a major part of SYTO 13 fluorescence. The fluorescence cannot be considered as a direct reflection of nucleic acid content. Other factors, such as topology or supercoiling, need to be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the efficacy of SYTO 13 for labelling bacteria and for assessing the distinct physiological status. A better knowledge of the parameters implicated in its fluorescence emission has been achieved.

Cell Cycle↗
PubMed2006

Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo.

Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.

Adaptation, Physiological↗
PubMed1986

Cloning and nucleotide sequence of the Bacillus megaterium gene coding for small, acid-soluble spore protein B.

The Bacillus megaterium gene coding for small, acid-soluble spore protein (SASP) B was cloned and its nucleotide sequence was determined. The amino acid sequence predicted from the DNA sequence was identical to that determined previously for SASP B, with the exception of the amino-terminal methionine predicted from the gene sequence which is presumably removed posttranslationally and an asparagine residue predicted at position 21 which was originally identified as an aspartate residue. The mRNA encoded by the SASP B gene is synthesized for only a discrete period midway in sporulation, in parallel with mRNAs coding for other SASPs. The small size of the SASP B mRNA (365 nucleotides) indicated that the mRNA is monocistronic. The SASP B gene itself hybridized strongly to only one band in Southern blots of restriction enzyme digests of B. megaterium DNA, suggesting that the SASP B gene is not a member of a highly conserved multigene family, as is the case for other SASP genes.

Amino Acid Sequence↗
PubMed1999

Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR.

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.

ATP-Binding Cassette Transporters↗
PubMed2004

Virulence gene expression in vivo.

The ability to identify and isolate bacterial RNA from animals or humans with infections has markedly advanced the capacity to examine microbial gene expression in vivo. This advance has been coupled with the development of quantitative real-time reverse transcription polymerase chain reaction and expression microarrays to allow investigators to accurately measure how organisms are manipulating their genetic expression during actual infections. Though the full ramifications of these technologies have yet to be realized, they promise to open new avenues of therapeutics for a broad range of infectious diseases by allowing researchers to focus on in vivo expressed genes. These developments provide a framework for efficient utilization of the vast amount of information being generated by the accelerating pace of genomic sequencing of microbes.

Animals↗
PubMed2005

The NusA:RNA polymerase ratio is increased at sites of rRNA synthesis in Bacillus subtilis.

Bacterial RNA polymerases (RNAPs) are capable of producing full-length transcripts in the absence of additional factors using in vitro assays. However, in vivo RNAP can become stalled during the elongation phase of transcription due to the presence of various sequence motifs. Subsequently, a host of elongation factors are required to modulate the activity of RNAP. NusA, the most intensively studied elongation factor, plays a role in increasing RNAP pausing and termination. Conversely, it is also important in transcription of rRNA where it functions as an anti-termination factor, helping to ensure only full-length transcripts are produced. Here we show that NusA is closely associated with RNAP within the bacterial nucleoid and that it is preferentially recruited to sites of rRNA synthesis. In vivo and in vitro analyses indicate this results in a change in stoichiometry of NusA:RNAP from 1:1 to approximately 2:1 at the subcellular sites of rRNA synthesis. A model is presented showing how the ratio of NusA:RNAP could affect the activity of the elongation complex so that it functions as an anti-terminator complex during rRNA synthesis.

Bacillus subtilis↗
PubMed2001

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Amino Acid Sequence↗
PubMed1983

[Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat].

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.

Adenosine Triphosphate↗
PubMed2004

Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, sigma-subunit and GreB.

Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs. The process of transcription is accompanied by complex structural rearrangements of RNAP. Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP. Using in vitro selection methods, we obtained 13 classes of ssDNA aptamers against Escherichia coli core RNAP. The minimal nucleic acid scaffold (an oligonucleotide construct imitating DNA and RNA in elongation complex), rifampicin and the sigma70-subunit inhibited binding of the aptamers to RNAP core but did not affect the dissociation rate of preformed RNAP-aptamer complexes. We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel. In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP-aptamer complexes. This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP-aptamer complexes by inducing conformational changes in the channel. We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investigating the conformational flexibility of the enzyme.

Base Sequence↗
PubMed2006

Antibacterial lysine analogs that target lysine riboswitches.

Lysine riboswitches are bacterial RNA structures that sense the concentration of lysine and regulate the expression of lysine biosynthesis and transport genes. Members of this riboswitch class are found in the 5' untranslated region of messenger RNAs, where they form highly selective receptors for lysine. Lysine binding to the receptor stabilizes an mRNA tertiary structure that, in most cases, causes transcription termination before the adjacent open reading frame can be expressed. A lysine riboswitch conceivably could be targeted for antibacterial therapy by designing new compounds that bind the riboswitch and suppress lysine biosynthesis and transport genes. As a test of this strategy, we have identified several lysine analogs that bind to riboswitches in vitro and inhibit Bacillus subtilis growth, probably through a mechanism of riboswitch-mediated repression of lysine biosynthesis. These results indicate that riboswitches could serve as new classes of antibacterial drug targets.

Anti-Bacterial Agents↗
PubMed1987

Defective degradation of bacterial DNA by phagocytes from patients with systemic and discoid lupus erythematosus.

The digestion of bacterial DNA by peripheral blood monocytes was impaired both in patients with systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE). The monocytes of these patients had both a small quantitative defect in the solubilization of DNA and a marked qualitative defect in the extent to which this DNA was degraded. In addition, neutrophils from patients with SLE released significantly less high molecular-weight DNA than control cells. Digestion of bacterial RNA and protein by phagocytes was not defective in either disease. The reduced digestion of DNA by phagocytes resulted in concomitantly larger amounts of high molecular-weight DNA remaining in these cells. Such sequestration of DNA may contribute to the persistence of fairly large DNA fragments in the tissue of patients with lupus erythematosus.

Adult↗