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PubMed1967

Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA.

Nucleotide sequences of 50 RNA codons recognized by amphibian and mammalian liver transfer RNA preparations were determined and compared with those recognized by Escherichia coli transfer RNA. Almost identical translations were obtained with transfer RNA from guinea pig liver, Xenopus laevis liver (South African clawed toad), and E. coli. However, guinea pig and Xenopus transfer RNA differ markedly from E. coli transfer RNA in relative response to certain trinucleotides. Transfer RNA from mammalian liver, amphibian liver, and amphibian muscle respond similarly to trinucleotide codons. Thus the genetic code is essentially universal, but transfer RNA from one organism may differ from that of another in relative response to some codons.

Amino Acid Sequence↗
PubMed1970

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes.

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A(7) (GA(7)), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA(7) also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA(7) solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.Age of the seedling tissue had a profound influence on the chromatin characteristics. With progression of development from the 2-day to the 4-day stage, the endogenous chromatin activity declined while the DNA template activity increased.

Journal Article↗
PubMed1998

Recognition of a human arrest site is conserved between RNA polymerase II and prokaryotic RNA polymerases.

DNA sequences that arrest transcription by either eukaryotic RNA polymerase II or Escherichia coli RNA polymerase have been identified previously. Elongation factors SII and GreB are RNA polymerase-binding proteins that enable readthrough of arrest sites by these enzymes, respectively. This functional similarity has led to general models of elongation applicable to both eukaryotic and prokaryotic enzymes. Here we have transcribed with phage and bacterial RNA polymerases, a human DNA sequence previously defined as an arrest site for RNA polymerase II. The phage and bacterial enzymes both respond efficiently to the arrest signal in vitro at limiting levels of nucleoside triphosphates. The E. coli polymerase remains in a template-engaged complex for many hours, can be isolated, and is potentially active. The enzyme displays a relatively slow first-order loss of elongation competence as it dwells at the arrest site. Bacterial RNA polymerase arrested at the human site is reactivated by GreB in the same way that RNA polymerase II arrested at this site is stimulated by SII. Very efficient readthrough can be achieved by phage, bacterial, and eukaryotic RNA polymerases in the absence of elongation factors if 5-Br-UTP is substituted for UTP. These findings provide additional and direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough mechanisms.

Base Sequence↗
PubMedDate not supplied

Bacterial small RNA regulators.

Small regulatory RNAs can modify the activity of proteins and the stability and translation of mRNAs. They have now been found in a wide range of organisms, and can play previously unsuspected critical regulatory roles. The bacterial small RNAs include two major classes. The largest family(with at least 20 members in Escherichia coli K12) acts by base pairing with target mRNAs to modify mRNA translation or stability; this class of RNAs also uses an RNA chaperone protein, Hfq. DsrA is the best-studied example of this family of RNAs. It has been shown to positively regulate translation of the transcription factor RpoS by opening an inhibitory hairpin in the mRNA, and to negatively regulate translation of hns by pairing just beyond the translation initiation codon. The class of RNAs that modify activity of proteins is exemplified by CsrB and CsrC of E. coli, two RNAs that bind to and inhibit CsrA, a protein translational regulator. Homologs of CsrA and related regulatory RNAs have been implicated in the regulation of gluconeogenesis, biofilm formation,and virulence factor expression in plant and human pathogens.

Bacterial Outer Membrane Proteins↗
PubMed2002

Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA.

In reactive and postinfectious arthritis the joints are generally sterile but the presence of bacterial antigens and nucleic acids has been reported. To investigate whether organisms traffic to affected joints in these conditions, we performed reverse transcription PCR using universal primers to amplify any bacterial 16S rRNA sequences present in synovial fluid. Bacterial sequences were detected in most cases, even after treatment of the synovial fluid with DNase, implying the presence of bacterial RNA and therefore of transcriptionally active bacteria. Analysis of a large number of sequences revealed that, as reported in rheumatoid arthritis, most were derived from gut and skin commensals. Organisms known to have triggered arthritis in each case were not found by sequencing the products obtained using universal primers, but could in some cases be shown to be present by amplifying with species specific primers. This was the case for Yersinia pseudotuberculosis and Chlamydia trachomatis. However, in arthritis thought to be related to Campylobacter infection the sequences obtained were not from Campylobacter jejuni or C. coli, but from other Campylobacter spp. that are not known to be associated with reactive arthritis and are probably present as commensals in the gut. We conclude that although rRNA from reactive arthritis associated organisms can be detected in affected joints, bacterial RNA from many other bacteria is also present, as was previously noted in studies of other forms of inflammatory arthropathy.

Arthritis↗
PubMed2003

Cutaneous manifestations of Crohn's disease, its spectrum, and its pathogenesis: intracellular consensus bacterial 16S rRNA is associated with the gastrointestinal but not the cutaneous manifestations of Crohn's disease.

The classic pathology of skin disease discontinuous from the inflamed gastrointestinal (GI) tract in patients with Crohn's disease (CD) includes pyoderma gangrenosum (PG), erythema nodosum (EN), and so-called metastatic Crohn's disease. The purpose of this study was two-fold: First, we explored the full spectrum of cutaneous lesions associated with Crohn's disease, and second, we sought to explore a potential molecular basis of the skin lesions in patients with CD. In this regard, we analyzed skin and GI tract biopsies from affected patients for the consensus bacterial SrRNA to determine whether direct bacterial infection was associated with either condition. Formalin-fixed, paraffin-embedded sections were studied and correlated to clinical presentation and histories from 33 patients with CD. Consensus bacterial RNA sequences were analyzed using an RT in situ PCR assay on both skin biopsy and GI biopsy material. The GI tract material included biopsies from 3 patients who had skin lesions and from 7 patients in whom there were no known skin manifestations. There were 8 cases of neutrophilic dominant dermal infiltrates, including pyoderma gangrenosum, 6 cases of granuloma annulare/necrobiosis lipoidica-like lesions, 5 cases of sterile neutrophilic folliculitis, 5 cases of panniculitis, 4 cases of vasculitis, 2 cases of psoriasis, 2 cases of lichenoid and granulomatous inflammation, and 1 case of classic metastatic CD. Intracellular bacterial 16S rRNA was detected in 8 of 10 tissues of active CD in the GI tract, of which 3 of the cases tested were from patients who also developed skin lesions at some point in their clinical course; in contrast, none of the skin biopsies had detectable bacterial RNA. The dermatopathological manifestations of CD discontiguous from the involved GI tract mucosa have in common a vascular injury syndrome, typically with a prominent extravascular neutrophilic and/or histiocytic dermal infiltrate. In addition, this study, the first to document in situ intracellular consensus bacterial SrRNA in the GI tract in CD, suggests that hematogenous dissemination of viable microbes is not associated with the cutaneous manifestations of this disease. Bacteria do, however, appear to play a role in bowel lesions of patients with CD.

Adolescent↗
PubMed1998

Core-sigma interaction: probing the interaction of the bacteriophage T4 gene 55 promoter recognition protein with E.coli RNA polymerase core.

The bacterial RNA polymerase sigma subunits are key participants in the early steps of RNA synthesis, conferring specificity of promoter recognition, facilitating promoter opening and promoter clearance, and responding to diverse transcriptional regulators. The T4 gene 55 protein (gp55), the sigma protein of the bacteriophage T4 late genes, is one of the smallest and most divergent members of this family. Protein footprinting was used to identify segments of gp55 that become buried upon binding to RNA polymerase core, and are therefore likely to constitute its interface with the core enzyme. Site-directed mutagenesis in two parts of this contact surface generated gene 55 proteins that are defective in polymerase-binding to different degrees. Alignment with the sequences of the sigma proteins and with a recently determined structure of a large segment of sigma70 suggests that the gp55 counterpart of sigma70 regions 2.1 and 2.2 is involved in RNA polymerase core binding, and that sigma70 and gp55 may be structurally similar in this region. The diverse phenotypes of the mutants implicate this region of gp55 in multiple aspects of sigma function.

Bacteriophage T4↗
PubMed2004

A bacterial two-hybrid system based on transcription activation.

We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription activation and involves the synthesis of two fusion proteins within the bacterial cell whose interaction stimulates transcription of a reporter gene. Specifically, one of the fusion proteins can function as a transcription activator when its interaction partner is fused to a subunit of the bacterial RNA polymerase. This bacterial two-hybrid system has been used to study a number of interacting proteins from both prokaryotes and eukaryotes, and can be used to find interacting proteins from complex protein libraries.

DNA-Directed RNA Polymerases↗
PubMed1978

Synthesis and properties of O6-methyldeoxyguanylic acid and its copolymers with deoxycytidylic acid.

This paper describes the synthesis of O6-methyldeoxyguanosine triphosphate (m6dGTP) and its copolymerization to high molecular weight polymer with deoxycytidylic acid. The monomer, m6dGTP, was synthesized from deoxyguanosine first protected by acetylation of the sugar hydroxyls, and then chlorinated in the 6-position with POCl3. The product, 6-chloro-3',5'-di-O-acetyl deoxyguanosine, was converted to O6-methyldeoxyguanosine with sodium methoxide and phosphorylated in the 5' position with carrot phosphotransferase. Monophosphate was converted chemically to the triphosphate and copolymerized with dCTP by terminal deoxynucleotidyl transferase. The resulting template, which contained O6-methylguanine, was tested for its ability to direct RNA synthesis by bacterial RNA polymerase. The presence of O6-methylguanine was shown to lead to the misincorporation of UMP in the product polymer, thus strengthening the hypothesis that O6-methylguanine is a promutagenic base.

Deoxycytidine Monophosphate↗
PubMed1973

Transcription of rat-liver chromatin with homologous enzyme.

The product of transcription of rat-liver chromatin with homologous rat-liver Form-B polymerase in vitro is high molecular weight RNA with sedimentation coefficients principally in the range of 18-45 S. The average size is somewhat smaller than that of the heterogeneous high molecular weight RNA synthesized by the endogenous enzyme in vivo (or in isolated nuclei). We have excluded the possibility that this difference can be attributed to degradation of the nascent RNA occurring under the conditions of our experiments. The size of the RNA produced by the homologous enzyme nevertheless approaches that of the natural transcripts much more closely than does that of the RNA produced by bacterial RNA polymerases, which we, in addition to other authors, have found to sediment around 10 S.

Animals↗
PubMed2003

Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs.

Polyadenylation plays an important role in RNA degradation in bacterial cells. In Escherichia coli, exoribonucleases, mostly RNase II and polynucleotide phosphorylase, antagonize the synthesis of poly(A) tails by poly(A) polymerase I (PAP I). In accordance with earlier observations showing that only a small fraction of bacterial RNA is polyadenylated, we demonstrate here that approximately 10% of rpsO mRNA harbors short oligo(A) tails ranging from one to five A residues in wild-type cells. We also compared the length, frequency and distribution of poly(A) tails at the 3'-end of rpsO transcripts in vivo in the presence and absence of Hfq, a host factor that in vitro stimulates the activity of PAP I, and found that Hfq affects all three parameters. In the hfq(+) strain the average length of oligo(A) tails and frequency of polyadenylated transcripts was higher than in the hfq(-) strain and a smaller proportion of tails was found at the 3' end of transcripts terminated at the Rho- independent terminator. Our data led us to the conclusion that Hfq is involved in the recognition of 3' RNA extremities by PAP I.

3' Untranslated Regions↗
PubMed2006

RNA-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of RNA polymerase by the Q antiterminator.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation, the stability of interactions between sigma(70) (the primary sigma factor in Escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent RNA. Here we demonstrate that the nascent RNA-mediated destabilization of an interaction between sigma(70) region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation. We demonstrate further that the requirement for nascent RNA in the process by which Q engages RNAP can be bypassed if sigma(70) region 4 is removed. Our findings illustrate how a regulator can exploit the nascent RNA-mediated reconfiguration of the holoenzyme to gain access to the enzyme during early elongation.

Base Sequence↗
PubMed2005

Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell.

The hepatitis C virus (HCV) is a major etiological agent causing chronic hepatitis in humans. Since the virus does not grow in a cell culture, the direct measurement of viral replication is impossible. Therefore, the current study presents a surrogate model system using a viral polymerase and RNA template. A plasmid expressing the HCV NS5B polymerase was maintained with a plasmid containing a reporter gene in an Escherichia coli cell. The reporter construct contained the HCV 5' untranslated region (UTR) followed by a luciferase gene with a specific orientation so that a minus-sense transcript containing the luciferase fused to the 5' UTR was produced after the initial transcription. When the HCV NS5B polymerase was expressed in the same cell, the primary transcript was recognized by the polymerase due to the presence of the minus-sense 5' UTR, and a secondary transcript containing a plus-sense luciferase gene was produced. Thus, a simple luciferase assay was able to measure the HCV NS5B polymerase activity. The production of minus- and plus-sense transcripts was confirmed by an RT-PCR, while the production of HCV NS5B and expression of the reporter luciferase in the bacterial cell were confirmed by immunofluorescence microscopy. The polymerization occurred in the absence of any other viral/host factors. Accordingly, this would appear to be the first study to demonstrate that the heterologous expression of an animal viral RNA polymerase and its template in a bacterial cell can partially reconstitute the polymerization reaction.

Animals↗
PubMed1988

Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.

The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities. We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE). Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes. The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies. The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis. In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259: 14880-14887). From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

Amino Acid Sequence↗
PubMed1988

Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp.

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.

Chloramphenicol↗