A hybridization assay specific for ribosomal RNA from Escherichia coli

Summary Ribosomal RNA fromEscherichia coli forms specific hybrids with heterologous DNA fromVibrio metschnicovii. The conditions for such hybridization are described, in which the amount of mRNA fromE. coli binding to theVibrio DNA is about 3% of the amount which hybridizes to the same amount of DNA fromE. coli.     

Structures of small subunit ribosomal RNAsin situ fromEscherichia coli andThermomyces lanuginosus

Small ribosomal subunits from the prokaryoteEscherichia coli and the eukaryoteThermomyces lanuginosus were imaged electron spectroscopically, and single particle analysis used to yield three-dimensional reconstructions of the net phosphorus distribution representing the nucleic acid (RNA) backbone. This direct approach showed both ribosomal RNAs to have a three domain structure and other characteristic morphological features. The eukaryotic small ribosomal subunit had a prominent bill present in the head domain, while the prokaryotic subunit had a small vestigial bill. Both ribosomal subunits contaied a thick collar central domain which correlates to the site of the evolutionarily conserved ribosomal RNA core, and the location of the majority of ribosomal RNA bases that have been implicated in translation. The reconstruction of the prokaryotic subunit had a prominent protrusion extending from the collar, forming a channel approximately 1.5 nm wide and potentially representing a bridge to the large subunit in the intact monosome. The basal domain of the prokaryotic ribosomal subunit was protein free. In this region of the eukaryotic subunit, there were two basal lobes composed of ribosomal RNA, consistent with previous hypotheses that this is a site for the non-conserved core ribosomal RNA.     

A membrane-derived 12-kD protein fromEscherichia coli with specific binding to the replication origin

A 12-kD protein, isolated from the membrane ofEscherichia coli, binds specifically to the 1.8-kb region containing the replication origin,oriC. It has two attachment sites within the 526-bporiC DNA fragment (HinfI3977-PstI1488), one of which is located in the positionHinfI3977-BglII22 and the other between base pairs 244 and 417.     

Characterization of an RNA "binding site" for a specific ribosomal protein of Escherichia coli

Summary A portion of the 16S ribosomal RNA that binds specifically to, and is protected from nucleolytic attack by, ribosomal protein S4 has been characterized in terms of its partial primary structure. The specific RNA (S4aR) in question comprises slightly more than onefourth of the full 16S molecule, and appears to be located (at least in part) in the 5-proximal half of the molecule. The functional significance of S4aR is discussed.     

A continuous spectrophotometric assay for Escherichia coli alanyl-transfer RNA synthetase

A new continuous spectrophotometric assay is demonstrated for Escherichia coli alanyl-tRNA synthetase. It involves β-γ adenylyl imidophosphate as a substitute for ATP in the pyrophosphate exchange reaction. The net conversion of β-γ adenylyl imidophosphate to ATP can be linked to NADP reduction by hexokinase and glucose-6-P dehydrogenase catalyzed reactions, which can be monitored at 340 nm. This assay can be extended to other aminoacyl-tRNA synthetases which can use β-γ nonhydrolyzable analogs of ATP as an ATP substitute.     

A catalytically inactive form of isocitrate dehydrogenase fromEscherichia coli

A protein exhibiting immunological cross-reactivity with NADP-specific isocitrate dehydrogenase, but containing no catalytic activity, has been isolated from nalidixic acid-resistantEscherichia coli. The two proteins have, within the limits of experimentation, identical molecular weight, subunit structure, and amino acid homology. The absence of catalytic activity in the protein isolated from nalidixic acid-resistant mutants may result from a mutation in the isocitrate dehydrogenase structural gene.     

Cloning of a gene coding for phosphotransacetylase fromEscherichia coli

Summary A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library ofEscherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities ofpta ⁺ plasmid-containing strains were amplified about 150-fold under control of thelac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thepta gene was found to be downstream ofackA by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of thepurF-folC-hisT region of the chromosome.     

Control of ribosomal RNA synthesis in Escherichia coli. III. Cytoplasmic factors for ribosomal RNA synthesis.

The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the "4S" fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation or rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acid-starved rel+ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.     

Shape of phosphofructokinase fromEscherichia coli in solution

Summary Small angle X-ray scattering measurements and electron microscopic studies were carried out onE. coli phosphofructokinase (E.C. 2.7.1.11; ATP: D-fructose-6-phosphate-1-phosphotransferase). The results suggest a tetrahedral arrangement of the protomers resulting in a radius of gyration of the enzyme of R=34.6 Å and a Stokes' radius of R₀=44.0 Å. The stereochemical arrangement of the four protomers, each of a molecular weight of 35,000, within theE. coli enzyme was further substantiated by a comparison of theoretical scattering functions with the experimental scattering measurements in dilute solutions of phosphofructokinase under physiological conditions. Moreover, from other hydrodynamic measurements,e.g., intrinsic viscosity and sedimentation coefficient, theMandelkern-Scheraga factor, , was calculated to be 2,095×10⁶, which is significantly lower than the ₀ for rigid spheres of 2,112×10⁶. This low -value might be due to a considerable porosity of the four protomers for mobile water molecules. The -value of 2,095×10⁶ is an indication of a porous sphere of almost uniform density at aDebye shielding ratio of 6.5, corresponding to a sphere radius of 22.0 Å for one protomer and an inverse hydrodynamic shielding length of 0.45 Å^–1.Fachrichtung Biochemie der Pflanzen undFachrichtung Feinstrukturforschung und Elektronenmikroskopie.     

Chromosomal loci for 16S ribosomal RNA in Escherichia coli

Summary Genetic loci for 16S ribosomal RNA (rRNA) on the Escherichia coli chromosome were determined using the K-sequence, a characteristic oligonucleotide of strain K12, as a genetic marker. Oligonucleotide analyses of 16S rRNA from various recombinants between strain K12 and strain B(H) showed that the loci for 16S rRNA containing the K-sequence were near the metB locus which was at 77 min. on the chromosome map.     

99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

PurposeRadiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.Methods and resultsIdentical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were ^99mTc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of ^99mTc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8 pmol, respectively, per 10¹⁰ cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90 min for both ^99mTc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of ^99mTc-study MORF was significantly higher (p = 0.009) than that of the control in the thigh with sterile inflammation.ConclusionA ^99mTc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.     

Characterization of adenylate cyclase fromEscherichia coli

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains ofEscherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0 –9.5. The enzyme has an absolute requirement for Mg²⁺ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.     

A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization

A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.     

A new mutation affecting ribosomal RNA synthesis in Escherichia coli.

A temperature-sensitive mutant of Escherichia coli is described. At the nonpermissive temperature there is a 12-fold reduction in the rate of rRNA synthesis, while tRNA and mRNA syntheses are affected to only a slight extent. Both protein and DNA syntheses also continue at nearly the normal rate. The mutation appears to affect the synthesis of 16S and 23S rRNA equally and has no detectable affect on rRNA maturation. The temperature-sensitive lesion appears to be caused by a single point mutation lying between minutes 21 and 27. It is suggested that this mutation seems to define a new genetic locus involved in the regulation of rRNA synthesis.     

In situ hybridization of iodinated ribosomal RNA to human chromosomes

Iodinated ribosomal RNA was hybridized to human metaphase chromosomes in a test of the effectiveness of iodinated products for in situ hybridization studies in a diploid system. The results indicate that ¹²⁵I is a feasible alternative as a source of radioactivity for autoradiographic mapping studies.     

Heavy meromyosin decoration of filaments fromEscherichia coli

A fibrous protein complex extracted fromEscherichia coli B/r by the method of Minkoff and Damadian [2] demonstrates arrowhead complexes when reacted with heavy meromyosin.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.