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At least 109 records · Page 6Linked to original sources
PubMed2003

Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase.

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

Animals↗
PubMedDate not supplied

[Effect of rifampicin on the synthesis of bacterial RNA polymerase mRNA by means of hybrid plasmids].

We studied the rate of synthesis of beta- and beta'-subunits of DNA-dependent RNA polymerase and the rate of beta-polypeptide mRNA synthesis in rifampicin-treated bacteria. The antibiotic doses used did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It is found that low doses of rifampicin cause an absolute and differential increase in the rate of synthesis of the specific mRNA for the beta-subunit, suggesting a stimulation of the corresponding gene transcription. However the absolute transcription stimulation does not fully correlate with the relative acceleration of beta-mRNA and the corresponding polypeptide synthesis. The stimulating effect of rifampicin on the beta-polypeptide synthesis was demonstrated also in a coupled system of transcription and translation directed by lambda rifd 47 DNA. The possible mechanisms of the rifampicin action are discussed.

DNA-Directed RNA Polymerases↗
PubMed1980

Specificity of promoter site utilization in vitro by bacterial RNA polymerases on Bacillus phage phi 29 DNA. Transcription mapping with exonuclease III.

Bacillus subtilis RNA polymerase holoenzyme transcribes phi 29 DNA in vitro producing five major RNA species defined by characteristic electrophoretic mobilities. In addition to these products, Escherichia coli RNA polymerase transcribes phi 29 DNA to yield three RNA species not detected when transcribing with the B. subtilis enzyme under the same optimal reaction conditions for RNA synthesis. Transcriptional analysis of purified restriction fragments and exonuclease III-digested DNA established locations of six promoter and three termination sites defining the eight transcripts. The transcription map shows that E. coli RNA polymerase initiates transcription at three sites not efficiently utilized by the B. subtilis enzyme. However, initiation by the B. subtilis polymerase from at least two of these sites could be detected at E:DNA ratios greater than 10 in the absence of competing promoters. These results indicate that differences between the two polymerases in promoter utilization are not explained by specificity of promoter binding, but represent differences in responding to promoter strength. Transcription of phi 29 DNA and T7 DNA by E. coli core polymerase with either B. subtilis or E. coli sigma subunits results in formation of transcripts identical with those produced by E. coli holoenzyme, suggesting that core polymerase contains elements important in determining relative promoter strength. The efficiency of rifampicin-resistant complex formation on phi 29 and T7 promoters is also dependent upon the source of core polymerase.

Bacillus subtilis↗
PubMed1976

[Biochemical determination of B. thuringiensis thermostabile, exotoxin, using the inhibition of bacterial RNA polymerases].

This method can determine amounts of exotoxin near 1 mug. Used on the autoclaved filtrates of cultures of B. thuringiensis var. thuringiensis (serotype 1 type-strain), it shows an excretion rate of 250 to 300 mug per millilitre. The results on different exotoxin-producer strains give a perfect parallelism with the results obtained by bio-assays on Insects, but the biochemical method is more accurate and reproducible. This biochemical technique allows us, for the first time, to place the exotoxin excretion during the sporulation stage and not during the exponential growth.

Bacillus thuringiensis↗
PubMed1967

Streptococcal nucleases. II. Characterization of DNAse D.

Preparations of streptococcal DNAse D with high specific activity and free of other streptococcal nucleases have been obtained by zone electrophoresis and column chromatography. Antisera prepared by injecting rabbits with such preparations specifically neutralize the activity of this enzyme. As with DNAse B, preparations of DNAse D regularly exhibit ribonuclease activity. For both B and D enzymes, the order of substrate preference is thymus DNA, yeast RNA, bacterial RNA; but the specific activity of the D enzyme is higher than that of the B enzyme with respect to thymus DNA and lower with respect to bacterial RNA. Both the deoxyribonuclease and the ribonuclease activities exhibited by preparations of both enzymes are inhibited by bacterial RNA, but approximately 100-fold greater concentrations of bacterial RNA are required to achieve inhibition of the deoxyribonuclease activity of the D enzyme equivalent to the inhibition of the B enzyme. The deoxyribonuclease activity of the D enzyme is also inhibited by yeast RNA, but even larger amounts are required. These observations indicate that the D enzyme is immunologically distinct from the other streptococcal nucleases and that it differs quantitatively from the B enzyme with respect to relative specific activities on different substrates and behavior in the presence of the bacterial ribonucleic acid inhibitor.

Citrates↗
PubMed2006

Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq.

SgrS is an Hfq-binding small antisense RNA that is induced upon phosphosugar stress. It forms a ribonucleoprotein complex with RNase E through Hfq to mediate silencing of the target ptsG mRNA encoding the membrane component of the glucose-specific phosphoenolpyruvate phosphotransferase system. Although SgrS is believed to act on ptsG mRNA through base pairing between complementary regions, this was not previously tested experimentally. We addressed the question of whether SgrS indeed forms an RNA-RNA duplex with ptsG mRNA to exert its regulatory function. Specific single nucleotide substitutions around the Shine-Dalgarno (SD) sequence of ptsG completely eliminated SgrS action while compensatory mutations in SgrS restored it. A systematic mutational analysis of both ptsG and SgrS RNAs revealed that six base pairs around SD sequence of ptsG are particularly important for SgrS action. We also showed in vitro that SgrS forms a stable duplex with the ptsG mRNA, and that Hfq markedly facilitates the rate of duplex formation.

Base Pairing↗
PubMed2003

Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.

Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant difference between PBC and AIH. The group of autoimmune liver diseases (PBC + AIH, N = 107, 29.9%) differed highly significantly from HC, chronic inflammatory bowel diseases (CD + UC + PSC/UC, N = 73, 6.8%), ALC, and HCV and also differed significantly (P = 0.01) from the group with bacterial and parasitic diseases (Yers + Camp + QF + MT, N = 86,13.95%) and from the group with Vasc + SLE (N = 68,13.2%). Testing of ARPA using the protein from E. coli yielded nearly identical results. In conclusion, an increased prevalence of antibodies to the beta-subunit of bacterial RNA polymerase, a highly conserved non-species-specific bacterial protein, can be found in primary biliary cirrhosis, but also in autoimmune hepatitis type I. These findings do not add an argument for a bacterial trigger of PBC. Rather, they suggest that ARPA belong to the pool of natural antibodies that are up-regulated in autoimmune liver diseases.

Antibodies, Bacterial↗
PubMed1988

A rapid method to quantitate non-labeled RNA species in bacterial cells.

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

Bacillus subtilis↗
PubMed1979

Stability of bacterial messenger RNA in mesophiles and thermophiles.

The decay of [3H]uridine-labeled mRNA was measured in the mesophile, Bacillus licheniformis (grown at 37 degrees C and 46 degrees C), and in the thermophile, Bacillus stearothermophilus (grown at 46 degrees C and 55 degrees C). For each organism, the half-life of the mRNA decreased as the growth temperature was increased. The stability index (half-life of mRNA/doubling time of cells), however, was remarkably constant for each organism regardless of the growth temperature. It is concluded that these results support the concept that kinetic considerations play a significant role in the explanation of thermophily.

Bacillus↗