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.     

Further mapping of 5S RNA cistrons in Escherichia coli

Summary Fine mapping data for four 5S RNA cistrons in E. coli are presented.     

Localization of some 5s RNA cistrons on Escherichia coli chromosome

Summary 3 5S RNA cistrons, characterized by specific nucleotides sequences, are shown not to be linked.     

Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli

A mutant of Escherichia coli with a partially defective phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase) has been characterized genetically. The genetic lesion causing the altered phosphoribosylpyrophosphate synthetase, prs, was mapped at 26 min on the linkage map by conjugation. Transductional analysis of the prs region established the gene order as purB-fadR-dadR-tre-pth-prs-hemA-trp. Two additional mutations were identified in the mutant: one in gsk, the gene encoding guanosine kinase, and one in lon, conferring a mucoid colony morphology. The contribution of each mutation to the phenotype of the mutant has been evaluated.     

In vitro synthesis of Escherichia coli ribosomal RNA

Synthesis of ribosomal RNA in a cell-free system was achieved using purified Escherichia coli RNA polymerase and bacterial DNA templates from E. coli, Proteus mirabilis and E. coli/P. mirabilis hybrid strains carrying an E. coli DNA enriched for ribosomal RNA genes.Both direct and indirect competition hybridization revealed that from 5 to 15% of the in vitro product, depending on the template used, had sequences homologous to rRNA. The level of synthesis of sequences homologous to rRNA was related directly to the proportion of rRNA genes in the template. The use of heterologous DNA during competition hybridization ensured at least a 100-fold greater sensitivity for the detection of rRNA sequences than from any messenger RNA sequence.     

Chromosomal location of the structural gene for glycerol kinase in Escherichia coli

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), and E. C. C. Lin. Chromosomal location of the structural gene for glycerol kinase in Escherichia coli. J. Bacteriol. 91:1763-1766. 1966.-A glycerol kinase mutant site has been mapped by transduction and sexual conjugation. Three-factor crosses with the two procedures yielded the following gene order: arginine-1-methionine-1-glycerol kinase-isoleucine, valine-16. An additional 13 independent glycerol kinase mutant sites mapped in the same region. Since some of the mutants were able to produce a protein serologically indistinguishable from the wild-type enzyme, it is concluded that the region mapped represents the structural gene for the kinase.     

Clustering of tRNA cistrons in Escherichia coli DNA

Characterization of tRNA:DNA hybrids reveals that many, perhaps most, of the tRNA genes in $\text{E. coli}$ DNA are clustered. Density and double-isotope measurements show that 3–4 molecules of tRNA can hybridize with DNA fragments that are only 4–5 times larger than a mature tRNA. Treatment of the hybrids with a single-strand-specific endonuclease results in the solubilization of 30–35% of the DNA and the formation of monocistronic hybrids.     

Nascent ribosomal and messenger RNA in DNA-membrane-complexes of Escherichia coli

Summary From Escherichia coli, DNA-membrane-complexes have been isolated which contain about 40% of the ribosomes, about 95% of the DNA and nearly all of the nascent RNA. The kinetic data on pulse labeled RNA show an average time of turnover of about 60 sec both for nascent messenger- and nascent ribosomal RNA. A proportion of the polysomes with nascent messenger RNA as well as most of the nascent ribosomal RNA is found in association with membranes, as has been shown by subfractionations of the DNA-membrane-complex involving treatment with DNase and desoxycholate. In this early transient stage, ribosomal precursor RNA already acquires some ribosomal proteins, as has been shown by arginine pulse label. Data on partial release of DNA from the DNA-membrane-complex by treatment with extremely low doses of DNase indicate that messenger RNA synthesis occurs in clusters on the DNA.The results support models in which, at any given time, RNA synthesis proceeds mainly in sections of the DNA close to the membrane. Thereby the DNA is linked to the membrane via nascent RNA contained in ribosomal precursors as well as via nascent messenger RNA on membrane-bound polysomes.     

Chromosomal location of the Escherichia coli cytochrome b556 gene, cybA

Summary The amounts of cytochrome b556 in the cytoplasmic membranes of several Escherichia coli K12 strains having F-prime factors and a lambda transducing phage were determined. The amount was amplified about two-fold in strains having F100-12 and F152, but not in strains having F100-11, F8 and psu ⁺ 2glnS ⁺. The strain TK3D11, which lacks the kdp-gltA region (deletion D-01) of the E. coli chromosome, did not synthesize cytochrome b556 at all. From these results, the gene cybA encoding cytochrome b556 was located in the kdp-gltA region.In the cytochrome b556-deficient mutant, a novel b type cytochrome, cytochrome b561 which is a product of the gene cybB, was identified. It seems to function as a physiological electron transferring cytochrome in place of cytochrome b556 in this mutant.Abbeviations HPLC high performance liquid chromatography - EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate - NADH reduced form of nicotinamide adenine dinucleotide     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.