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PubMed1979

The molecular topography of RNA polymerase-promoter interaction.

Ultraviolet irradiation forms covalent crosslinks between E. coli RNA polymerase and the lac UV5 promoter substituted with bromouracil in the place of thymine. I have determined the polymerase subunit and the base within the promoter sequence that are joined to each other in two such crosslinks. The sigma and beta subunits of RNA polymerase, respectively, are crosslinked to the third base upstream (-3) and the second base downstream (+3) from the starting point of transcription (+1). Both bases are on the nontemplate strand of the promoter DNA. The location of the beta subunit suggests that it forms at least part of the catalytic site of the enzyme. The disposition of sigma suggests that this subunit plays a direct role in unwinding the DNA at the promoter. The sigma crosslink is close to the "Pribnow Box," which is centered about 10 bases upstream from the RNA start site, contains a striking homology between promoters and is the locus of many promoter mutations.

Base Sequence↗
PubMed1978

Mutation affecting thermostability of sigma subunit of Escherichia coli RNA polymerase lies near the dnaG locus at about 66 min on the E. coli genetic map.

The Escherichia coli strain, ts-rnp5, originally described in 1975 by G. D. Burdick and H. Berger, is shown to possess an RNA polymerase (RNA nucleotidyltransferase) sigma subunit with an activity 4--6 times less thermostable at 45 degrees than sigma from wild-type strains. This defect remains associated with the sigma polypeptide through a variety of purification stages, including renaturation of sigma after its elution from sodium dodecyl sulfate/polyacrylamide gels. The mutation responsible for decreased thermostability of sigma, called rpoD1, cotransduces with dnaG and therefore is located at about 66 min of the E. coli genetic map.

Chromosome Mapping↗
PubMed1978

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

A gene affecting the sigma subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 min, and the order tolC-dnaG-sigma-tet was determined. We used frequency of contransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the sigma subunit. One such mutant was found to map at the sigma locus. The sigma subunit isolated from this mutant is unstable at 46 degrees C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is sigma dependent.

Chromosome Mapping↗
PubMed1979

Isolation and characterization of transducing phage coding for sigma subunit of Escherichia coli RNA polymerase.

A transducing phage has been isolated with codes for the sigma subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage lambda vector that is capble of forming lyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the sigma gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and sigma (rpoD) whereas those containing the Sac I fragment complement only sigma. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/translation system suggest that a Sac I site separates the promoter for sigma from the sigma structural gene. The direction of transcription of sigma was determined to be clockwise with respect to the E. coli genetic map.

Bacterial Proteins↗
PubMed1977

Effect of salt on the transcription of T7 DNA by RNA polymerase from T4 phage-infected E.coli.

Transcription of T7 DNA by T4 core enzyme with host sigma is more sensitive to KCI than that by host core enzyme with host sigma. When salt is added after initiation of RNA chains has occurred, it is not inhibitory. Salt affects the binding of T4 enzyme to T7 DNA to the same degree as the binding of host enzyme. Active preinitiation complex formation is inhibited more by salt with the T4 enzyme and the inhibition is temperature-dependent.

Coliphages↗
PubMed1978

DNA-dependent RNA polymerase from Halobacterium halobium.

DNA-dependent RNA polymerase core enzyme was isolated from Halobacterium halobium. The purification is based on the finding that the enzyme is stable in 40% (v/v) glycerol, in the presence of 0.05 M MgCl2 and involves adsorption of contaminants to DEAE-cellulose, precipitation of the complex of polymerase with DNA by streptomycin sulfate, chromatography over Biogel and affinity chromatography over heparin-Sepharose or heparin-cellulose. The enzyme consists of four or five different subunits. The composition formula was estimated as (150000) (86000)2 (72000)2 (49000)3 or 2; there may be one or two different 49000-Mr subunits. RNA synthesis requires a template. Denatured DNA is more efficient than native DNA. The transcription of native DNA is specifically stimulated by the addition of a possibly sigma-like factor eluted from DEAE-cellulose. The fidelity of transcription is indicated by the absolute requirement for UTP besides ATP with poly[d(A-T)] as the template.

DNA-Directed RNA Polymerases↗
PubMed2025

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide.

Bacteria secrete extracellular membrane vesicles (EMVs). Physiological functions and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the details of the molecular basis of EMV biogenesis have not yet been fully elucidated. In our previous work, an N-terminus-substituted FAAV peptide labeled with nitrobenzoxadiazole (NBD; nFAAV5-NBD) was developed. This peptide can sense the curvature of a lipid bilayer and selectively bind to EMVs even in the presence of cells. Here, we applied nFAAV5-NBD to a genome-wide screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. We analyzed the transposon insertion sites in hyper- and hypo-vesiculation mutants and identified 16 and six genes, respectively, with a transposon inserted within or near them. Targeted gene-disrupted mutants of the identified genes showed that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase β-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, d-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study demonstrates the utility of a novel screening method using a curvature-sensing peptide for mutants with altered EMV productivity and provides information on the genes related to EMV production.IMPORTANCEConventional methods for isolation and quantification of extracellular membrane vesicles (EMVs) are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying various genes associated with EMV production of Shewanella vesiculosa HM13, which would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be valuable for EMV applications, such as secreting useful substances as EMV cargoes and producing artificially functionalized EMVs.

Shewanella↗
PubMed1975

Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis.

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage phie. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover, during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with phi DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phase (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.

Bacillus subtilis↗
PubMed2026

The SigD regulon of Mycobacterium abscessus determines cell envelope composition and antibiotic susceptibility.

A major determinant of the exceptional intrinsic resistance of M. abscessus is the lipid-rich cell envelope, yet the regulatory systems that remodel envelope-associated pathways remain poorly defined. Here, we determine the σD regulon in M. abscessus and establish its role in cell envelope homeostasis and intrinsic resistance to hydrophobic antibiotics. RNA-Seq analysis of a MabΔsigD mutant identified 447 differentially expressed genes, while ChIP-Seq mapped 72 σD binding sites and defined a conserved promoter motif (GTAACA/G-N16-CGAT). Using a combination of σD binding, motif orientation and expression data, we identified a core set of directly regulated genes, distinct from what was previously observed in M. tuberculosis, many of which encode proteins involved in envelope-associated functions. These include loci involved in trehalose polyphleate (TPP) biosynthesis, the antigen 85 complex and peptidoglycan remodeling enzymes. Deletion of sigD resulted in a significant reduction in TPPs in the cell envelope and an increase in ethidium bromide accumulation. Consistent with these changes, loss of σD selectively sensitized M. abscessus to hydrophobic antibiotics, including rifampicin and tigecycline. Deletion of mmpL10, which is required for transport of TPP precursors, recapitulated the drug sensitivity of MabΔsigD, implicating envelope composition as a key effector of the phenotype. Expression of the σD regulon further increased during starvation and in response to SDS, isoniazid, and ethambutol, mediated by degradation of RsdA, consistent with a role in stress-responsive envelope adaptation. Together, these findings demonstrate σD is active during logarithmic growth in rich media where it regulates the expression of envelope-associated genes that influence envelope permeability and basal level susceptibility to hydrophobic antibiotics; its activity further increases in response to cell envelope stress, presumably promoting envelope remodeling to counteract damage.

Regulon↗
PubMed1979

Anion transport in red blood cells. I. Chemical properties of anion recognition sites as revealed by structure-activity relationships of aromatic sulfonic acids.

The present study is concerned with the chemical factors that determine the inhibitory properties of reversible aromatic sulfonic acids on sulfate exchange system of human red blood cells. Two series of compounds were tested for inhibitory potencies: benzene sulfonic acid (BS) and 2,2'-disulfonic stilbene (DS) derivatives, each series with substituent groups such as Cl, OH, NH2, NO2, NNN, N-acetamido, and N-benzoamido. As judged by various kinetic criteria, all congeners of BS and DS appear to have common sites of action in the anion transport system. The range of inhibitory potencies, as defined by the concentration required to produce 50% inhibition (ID50), varied over a 10(4) range (ID50:2-50,000 microM). The degree of inhibition was correlated with two physicochemical properties of the substituent groups: (a) lipophilicity, as judged by the pi values (Hansch factor) of the groups; and (b) the electronic character, as judged by sigma values (Hammett factor) of the groups. Optimal correlations were obtained with a linear combination of the two factors. Based on the above structure-activity relationships and on a comparison between the inhibitory properties of congeners of BS and DS, we suggest that the microenvironment of substrate recognition sites bears a positive multipolar character and possesses functionally essential groups with electron donor capacity embedded in a hydrophobic area.

Anions↗
PubMed1979

Sigma subunit of Escherichia coli RNA polymerase affects the function of lambda N gene.

A new class of Escherichia coli mutants, referred to as grn, has been isolated by localized mutagenesis. These mutations affect the sigma subunit of DNA-dependent RNA polymerase (ribonucleoside 5'-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) by abolishing the expression of the lambda N gene, and they are closely lniked to dnaG in the order dnaG-grn-uxaA. Detailed study of one such mutant, grn1, yielded the following results: (i) grn1 is a single mutation and the mutant cell shows cold-sensitivity in growth; (ii) the Grn phenotype of the mutant can easily be suppressed by secondary mutations in the beta subunit gene of RNA polymerase; (iii) purified holoenzyme of RNA polymerase isolated from the mutant showed an altered salt-dependency in vitro, and the mixed reconstitution of the mutant with the wild-type subunits showed that the sigma subunit of the grn1 mutant is altered; (iv) lambda phage mutants (lambda grg), which overcome the grn mutation, can be classified into two groups, the "nin-deletion" and the "N-mutant" groups (both of these are also able to grow on the previously described groN mutant of Georgopoulos and nusAB of Friedman); (iv) the mutant polymerase transcribed 12S as well as 7S RNA from lambda DNA in the presence of the rho factor in vitro. These results indicate that the grn mutation alters the sigma subunit of RNA polymerase and that the sigma subunit participates in activating the N-mediated antitermination mode of lambda phage transcription.

Bacteriophage lambda↗
PubMed1978

Nitrosamine carcinogenicity: a quantitative Hansch-Taft structure-activity relationship.

Statistically significant correlations have been demonstrated between carcinogenic activity, water-hexane partition coefficients and electronic factors for an extensive series of N-nitroso compounds. Electronic factors were expressed by the Taft sigma* values of substituents on the carbon atoms alpha to the N-nitroso group. Such correlations indicate that transport of the carcinogen to its active site has an important effect on its potency. The correlations also implicate reactivity at the alpha-carbon in the determination of carcinogenic activity and point out various structural types which do not follow the general rule.

Animals↗
PubMedDate not supplied

The molecular structure of the free acid of guanosine 3',5'-cyclic monophosphate (cyclic GMP).

The structure of the free acid of guanosine 3',5'-cyclic monophosphate has been determined from three dimensional x-ray diffraction data collected with a GE XRD-490 automated diffractiometer. The molecule crystallizes in the orthorhobic space group P212121 with a = 17.953(17), b = 7.530(7), and c = 12.755(12) and Z = 4. The structure was solved by direct methods. The 969 independent data were defined by full matrix least-squares to a final agreement factor, RF, of .050 for FO greater than sigma (FO). The compound exists as a switterion in the solid state. N(7) of the base is protonated. The structure of the free acid is similar to that of the sodium salt. Both have the syn conformation. The puckering of the sugar rings is also similar. Small differences exist in the bond angles of the cyclic phosphate and furanose rings.

Cyclic GMP↗
PubMed2026

Proteomic signatures for sudden cardiac death and related intermediate phenotypes.

BACKGROUND: Novel markers for sudden cardiac death (SCD) are needed. OBJECTIVE: This study aimed to explore whether a protein risk score derived from a large-scale proteomics dataset improves risk prediction of SCD in the general population. METHODS: A total of 52,705 individuals with 1459 unique plasma protein measurements were included from the UK Biobank Pharma Proteomics Project. A protein risk score was developed using lasso-penalized Cox regression on 40,722 participants enrolled at the English centers and validated on 11,983 participants enrolled at the remaining centers. RESULTS: The protein risk score formula developed from the derivation set comprised 64 unique plasma proteins including latent-transforming growth factor beta-binding protein 2, protein tyrosine phosphatase receptor sigma, and spondin-1. In the test set, a per standard deviation increase in protein risk score was associated with a hazard ratio of 2.60 (95% confidence interval [CI] 2.12-3.18) for SCD. Adding a protein risk score to SCD clinical risk factors resulted in a concordance index increase of 0.063 (95% CI 0.037-0.105) for SCD. For ventricular arrhythmia-mediated SCDs, an increase in concordance index when a protein risk score was added to SCD clinical risk factors was 0.070 (95% CI 0.010-0.188). A protein risk score added to SCD clinical risk factors resulted in a risk reclassification of 16.9% (95% CI 9.0-24.7) at a 10-year risk threshold of 5%. A protein risk score was significantly associated with intermediate phenotypes of SCD including corrected QT prolongation, an increase in left ventricular mean myocardial thickness, and a decrease in left ventricular global longitudinal strain. CONCLUSION: A protein risk score derived from a single plasma sample significantly improved risk prediction of SCD and related intermediate phenotypes.

Humans↗
PubMed1976

RNA polymerase in vegetative cells of Bacillus subtilis. II. New polypeptide factors, FI and FII, stimulating in vitro RNA synthesis directed by phage M2 DNA.

Two polypeptides named FI and FII were isolated from vegetative cells of Bacillus subtilis Marburg. The molecular weights of FI and FII were 15,000 and 30,000 daltons, respectively. They were able to stimulate the transcription of phage M2 DNA in the presence of Mg2+ ions by RPase L1 and RPase L2 [RNA polymerase; EC 2.7.7.6]. Although both core- and holo-RPase L2 hardly exhibited transcription activity under these conditions, the factors could stimulate both activities up to the level of RPase L1 activity. The stimulation was much less marked when B. subtilis DNA was used as a template. These stimulatory functions were found to lie not in the chain elongation but in the initiation step of transcription, following the preinitiation step. To obtain stimulation by the factors, preincubation with RNA polymerase was necessary. FI stimulated RPase L1 or RPase L2 only when preincubated in the stimultaneous presence of FII, forming a complex, RPase L1(or L2)-FI-FII. On the other hand, FII alone could stimulate transcription, forming a complex. RPase L1 (or L2)-FII. In these complexes, the ratio of FI, FII, and RPase L1(or L2) was 1 : 1: 1. Although the core-RPase L2 activity was inhibited by sigma subunits, it was not inhibited by was rather stimulated when the enzyme was present as a complex with FI and FII. Thus the complex, consisting of RPase L2 and the factors, resembled RPase L1 with respect to molecular weight, template specificity, the effect of sigma subunit, and sensitivity to rifampicin.

Bacillus subtilis↗