Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO₄, CaCl₂, BaCl₂) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO₄ (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.     

Vesicular stomatitis virus infection reduces the number of active DNA-dependent RNA polymerases in myeloma cells.

Infection of mouse myeloma (MPC-11) cells with vesicular stomatitis virus resulted in rapid loss in activity of cellular RNA polymerases associated with nuclear chromatin. No RNA polymerase inhibitor could be detected in extracts of infected cell nuclei. Reconstitution experiments with solubilized RNA polymerases dissociated from chromatin of infected and uninfected cells demonstrated that vesicular stomatitis viral infection did not affect the ability of the polymerases to function on endogenous or exogenous templates; nor did infection alter the template capability of the chromatin. Measurement of the number of actively growing RNA chains revealed that infected cell nuclei contained fewer active polymerase units; however, the rates of RNA chain elongation were the same in nuclei from infected and uninfected cells. Quantitation of the number of polymerase units active in nuclear chromatin revealed that the alpha-amantin-sensitive polymerase II was more severely reduced by viral infection than were polymerases I and III.     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

Change in the Template Specificity of RNA Phage SP-Replicase by the <Emphasis Type="Italic">E. coli</Emphasis> Cell Component

Qβ-REPLICASE was isolated from E. coli infected with the RNA bacteriophage Qβ as RNA-dependent RNA polymerase which had template specificity¹. RNA phage SP², which is distinct from RNA phages isolated previously^3,4, has been isolated in our laboratory and SP-replicase⁵ was purified from E. coli infected with SP-phage. SP-replicase has a template specificity different from that of Qβ-replicase. By using this new RNA-replicase, comparison between two distinct replicases has become possible.     

Decoding phage resistance by mpr and its role in survivability of Mycobacterium smegmatis

Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the development of phage therapy. Hence, it is both essential and important to understand the mechanism of phage resistance in bacteria. First identified in Mycobacterium smegmatis, the gene mpr, upon overexpression, confers resistance against D29 mycobacteriophage. Presently, the mechanism behind phage resistance by mpr is poorly understood. Here we show that Mpr is a membrane-bound DNA exonuclease, which digests DNA in a non-specific manner independent of the sequence, and shares no sequence or structural similarity with any known nuclease. Exonuclease activity of mpr provides resistance against phage infection, but the role of mpr may very well go beyond just phage resistance. Our experiments show that mpr plays a crucial role in the appearance of mutant colonies (phage resistant strains). However, the molecular mechanism behind the emergence of these mutant/resistant colonies is yet to be understood. Nevertheless, it appears that mpr is involved in the survival and evolution of M. smegmatis against phage. A similar mechanism may be present in other organisms, which requires further exploration.     

Rhythmic changes in transcriptional activity during the development of potato tubers

Chromatin-bound, DNA-dependent RNA polymerase (EC 2.7.7.6) activity and chromatin template availability, as measured with saturating amounts of E. coli RNA polymerase, changes rhythmically during the formation, dormancy, and sprouting of potato tubers. Active growth processes coincide with the highest RNA polymerase activity as well as the greatest template accessibility, during tuberization and sprouting. Consequently, chromatin-associated RNA and protein content is highest in young developing tubers and in old tubers at the onset of sprouting. Ribosomal RNA content, in turn, is maximal in small tubers, remains constant during dormancy, and decreases when sprouting begins, probably due to the translocation of rRNA into the sprouts. The nucleolus changes its shape and size concomitantly with the process of tuberization.     

A new RNA synthesis mutant of E. coli

A temperature-sensitive mutant of E. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth of f2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1 away from rif ^r in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rif ^r T^s double mutants, the T ^s mutation suppresses rif ^r and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.This research was supported by Public Health Service grant GM-14368 from the National Institute of General Medical Sciences and by grant IN-29 from the American Cancer Society. One of us (D.P.) is a predoctoral trainee, supported by a National Science Foundation Graduate Traineeship Program and by a National Institutes of Health Predoctoral Research Fellowship. S. Marshall is supported by LASBAU.     

INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase

RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase.     

Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.