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At least 19 recordsLinked to original sources
PubMed1976

The 5' ends of bacterial RNA. II. The triphosphate-terminated ends of primary gene transcripts.

Bacterial RNA, Pulse labeled with 32Pi, was digested with pancreatic RNAase. Oligonucleotides containing a triphosphate group at the 5'-hydroxyl, and therefore derived from the original beginning ends of the RNA transcripts, were purified by hydroxyapatite chromatography and analyzed by two-dimensional paper electrophoresis. A broad diversity of species was found, although the distribution among these species was not completely uniform. Possible methods of utilizing these methods in combination with in vitro synthetic techniques are discussed.

Base Sequence↗
PubMed1976

Effect of nitrofurazone on bacterial RNA and ribosome synthesis and on the function of ribosomes.

Exposure of E. coli B/r to nitrofurazone strongly inhibits the synthesis of all classes of RNA and both ribosomal sub-units. Polysome formation is likewise inhibited. However, in E. coli nfr-207 a mutant of B/r which lacks nitrofurazone-reductase I, the synthesis of RNA, ribosomal sub-units and formation of polysomes are not significantly affected. This result implies that a reduced metabolite of the drug rather than the drug itself is the active agent. The ability of ribosomes isolated from nitrofurazone-treated E. coli B/r to carry out poly-U directed polyphenylalanine synthesis was lower than that of ribosomes from untreated cells. 14C from labelled nitrofurazone was found to bind to ribosomal sub-units.

DNA-Directed RNA Polymerases↗
PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗
PubMed1976

A bacterial RNA polymerase mutant that renders lambda growth independent of the N and cro functions at 42 degrees C.

We describe a bacterial RNA polymerase mutation, rif 501, which confers rifampicin resistance and thermosensitivity to E. coli K 12. The purified RNA polymerase enzyme from rif 501 bacteria shows increased heatsensitivity in vitro at 51 degrees C. However, in vivo, at 42 degrees C the non-permissive temperature, mutant bacteria continue to grow and to synthesize RNA for 90 min. On a lawn of the mutant bacteria, at 40-41 degrees C, phage lambda forms clear plaques (LycA phenotype); this is probably due to an enhancement of cro function; we surmise that at 42 degrees C the transcription originating from the pR (but not from the pL) promoter on the lamdba genome becomes N-independent and less sensitive to the absence of the cro product. We discuss the possibility that both the N and cro proteins of phage lambda interact directly with the bacterial RNA polymerase. These observations indicate that the loss of viability of the rif 501 mutant at the restrictive temperature is not a consequence of an immediate inactivation of RNA polymerase; rather we feel it is due to a modification of the activity of RNA polymerase, leading to a disruption of the cellular regulation.

Coliphages↗
PubMed1975

The 5'-termini of bacterial RNA. I. The monophosphorylated termini.

A two-dimensional fingerprint technique is described which separated oligonucleotides by length and number of phosphates present at the termini. The diversity of terminal nucleotide sequences was found to be quite restricted in bacterial 4-5 S RNA: in the strain used here the predominant dinucleotide species was pG-C, and the only trinucleotides produced were pG-G-Up and pG-G-Cp. Examination of total pulse-labeled RNA revealed the same restricted set of terminal trinucleotides.

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↗
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↗
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↗
PubMed1975

An intercistronic region and ribosome-binding site in bacterial messenger RNA.

A messenger RNA fragment about 220 nucleotides long has been isolated from 32-P-labeled tryptophan operon mRNA of Escherichia coli. When point mutations at the end of trpB and the beginning of trpA were introduced, the resulting nucleotide changes were found; hence the mRNA fragment must include the trpB-trpA intercistronic region. Most of the nucleotide sequences can be assigned to specific locations in the structural genes, based on the amino-acid sequences of the trpB and trpA proteins. In vitro, ribosomes bind to this piece of mRNA and protect from nuclease attack a region about 40 nucleotides long, containing a central AUG codon. The triplet codons to the 3' side of this AUG correspond to the first seven amino acids of the trpA protein; the codons to the 5' side correspond to the last six amino acids of the trpB protein. Translation of trpB is terminated by single UGA codon, which overlaps the trpA AUG initiation codon: UGAUG. Thus the untranslated "intercistronic" region consists of only two nucleotides. The RNA sequence spanning this region undoubtedly fulfills two functions, specifying ribosome recognition signals as well as encoding amino-acid sequences.

Base Sequence↗
PubMed1979

Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders.

The transcriptional properties of bacterial RNA polymerases purified from seven different species and representing a variety of bacterial orders have been studied using the well characterized DNA from phage T7 as template. The subunit composition of the different preparations suggests that each RNA polymerase holoenzyme has a promoter structure (betabeta'alpha2sigma) similar to that of the well studied E. coli and B. subtilis enzymes. Each enzyme utilizes DNA from bacteriophage T7 as an effective template for RNA synthesis, although all preparations contain a substantial fraction of inactive enzyme. Electrophoretic analysis of the RNA products made with the different RNA polymerases in vitro using T7 DNA (deletion mutant deltaD111) as template reveals that with minor exceptions, all of the heterologous RNA polymerases utilize the same collection of promoter sites on T7 used by the E. coli host enzyme, and only those promoter sites. The T7 early terminator is also efficiently utilized by each enzyme. Since the different bacterial species from which the RNA polymerases were derived are genetically quite distant, it appears that there is a structural element in the promoter which governs its recognition and which is universally recognized among RNA polymerases of different bacterial species. While the different bacterial RNA polymerases generally utilize the same set of T7 promoter sites, the efficiency of utilization of the different promoters varies considerably for different RNA polymerases and for different reaction conditions with the same RNA polymerase. Hence although the several T7 promoter sites share the ability to be recognized by bacterial RNA polymerases, each shows a unique pattern of utilization and therefore must possess a unique element of promoter structure as well. It has previously been shown (Stahl and Chamberlin, 1977) that T7 promoters A1, C, D and E interact differently with E. coli RNA polymerase as judged by the properties of complexes formed between each promoter and the latter enzyme. Since competition takes place among different promoter sites on a template and since these sites can differ functionally, small changes in reaction conditions or in the structure of the RNA polymerase can lead to significant changes in the rate of utilization of different promoter sites even when these promoter sites share common elements. Because the T7 promoters and terminator are utilized efficiently by such a wide range of RNA polymerases and because each of the several T7 promoters possesses unique properties which govern its utilization by RNA polymerase, analysis of the transcripts formed on a T7 DNA template provides a simple and rapid procedure for detecting and analyzing alterations in bacterial RNA polymerases which affect promoter or terminator recognition or utilization.

Bacteria↗
PubMed1977

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis.

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.

Amoeba↗
PubMed1975

Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3'-terminal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome.

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequences obtained were: Bacillus stearothermophilus, -G(Z)approximately 5 Y-U-C-C-U-U-U-C-U (A); B. subtilis, -G(Z)approximately 7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aerugionosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B stearothermophilus and B. subtilis, -Y-C; C. crescentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis.

Bacillus subtilis↗