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Biomedical subjects

F Imamoto

Publications and source records attributed to F Imamoto.

At least 91 records · Page 5Linked to original sources
PubMed1981

In vivo correlation between DNA supercoiling and transcription.

The superhelical density of pMT plasmid DNA in Escherichia coli cells was measured as a function of the transcriptional activity, which was reduced by treatment with coumermycin or oxolinic acid. Superhelicity was assayed by agarose gel electrophoresis of DNA extracted from cells gently lysed by sarkosyl. Coumermycin treatment reduced the proportion of supercoiled plasmid DNA in concert with a decrease in the rate of plasmid-coded synthesis of trp mRNA, implying a correlation between supercoiling of DNA and its suitability for transcription. On the other hand, the oxolinic acid-induced loss of supercoiled plasmid DNA was relatively small, while concomitant inhibition of trp mRNA synthesis was very severe. Treatment of the cells with these two drugs never removed all of supertwists from the pMT plasmids carried.

Aminocoumarins↗
PubMed1980

A biochemical assay for the transcription-antitermination function of the coliphage lambda N gene product.

An in vitro assay has been developed for the antiterminating activity of the N gene product (pN) of bacteriophage lambda, based on the N-dependent stimulation of trp mRNA synthesis from the DNA templates of lambda trp transducing phages. Using this assay system, we show (a) that the stimulation of trp mRNA synthesis by pN requires the cis-dominant nutL site on the lambda chromosome and (b) that pN can participate in vitro in the formation of functional antiterminating transcription complexes.

Bacteriophage lambda↗
PubMed1980

Purification and characterization of the N gene product of bacteriophage lambda.

The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coli protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing the nusA-nusB- mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.

Bacteriophage lambda↗
PubMed1979

Evidence for endonucleolytic cleavage at the 5'-proximal segment of the trp messenger RNA in Escherichia coli.

The 5'-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28.trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5'-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5' to 3' direction.

DNA, Viral↗
PubMed1979

In vivo enhancement of general and specific transcription in Escherichia coli by DNA gyrase activity.

The effect of drugs which inhibit DNA gyrase, including nalidixic acid, oxolinic acid and coumerycin, on transcription of Escherichia coli bacteria, phage and plasmid genomes was studied. Quantitative estimates of the synthesis of RNA under drug-treatment conditions showed that synthesis of many RNA species, including trp mRNA, was subject to inhibiton by the drug. Transcription directed by the lambda promoter pR was selectively less sensitive to the drug action than transcription initiated at the lambda promoter pL. Evidence was obtained showing that diminished transcription resulted from less frequent RNA chain initiation rather than a premature arrest of the chain elongation. Inhibiton of transcription by these DNA gyrase inhibitors was observed even in the absence of DNA replication. The inhibition by oxolinic acid or coumerycin was not observed in an E. coli strain bearing a nalAr mutation or a cour mutation, respectively. The reduction of trp mRNA synthesis in oxolinic acid-treated cells cannot be attributed to the increase in the rate of nascent mRNA degradation. These results indicate that DNA gyrase is generally required for intracellular RNA synthesis, and suggest that the supercoiling of DNA by this winding enzyme enhances the initiation of transcription.

Bacteriophage lambda↗
PubMed1978

Promoter-specific inhibition of transcription by antibiotics which act on DNA gyrase.

Nalidixic acid, novobiocin and coumermycin specifically inhibit phage promoter-dependent transcription of the trp operon in phi 80 ptrp but not transcription from the authentic trp promoter. The nalidixic acid inhibition is not observed in an E. coli strain containing a nalAr mutation. These results indicate that DNA gyrase is involved in transcription.

Coliphages↗
PubMed1978

Release of nascent trp mRNA from the operon DNA in chloramphenicol-treated cells of Escherichia coli.

When transcription of the E. coli trp operon is blocked as a result of inhibition of ribosomal activities by chloramphenicol, nascent trp mRNA but not RNA polymerase molecules seem to detach from the template DNA. The continued association of RNA polymerase with DNA is inferred because RNA synthesis resumes at or near the sites of transcription blockage after removal of the antibiotic. When E. coli cells infected with lambdatrp phage are incubated with chloramphenicol under the "Ptrp-promoted" conditions (Imamoto and Tani, 1972; Segawa and Imamoto, 1974), the trp mRNA molecules synthesized after removal of the antibiotic contain only promoter-distal information. They are usually small in size, in spite of the fact that chloramphenicol inhibition does not lead to the degradation of trp mRNA. On the other hand, PL-promoted trp transcription, which is non-polar and insensitive to chloramphenicol action, does not show such a premature release of nascent trp mRNA in chloramphenicol-treated cells.

Chloramphenicol↗
PubMed1978

Restoration of polarity by N-deficiency in lambda phage containing a translocated trp operon segment.

When translation of trp mRNA is terminated by a nonsense codon or by antibiotics like chloramphenicol, the amount of the mRNA distal to the blocked ribosomes is found at much lower levels ("polarity"). Polarity is alleviated when the trp mRNA is formed as part of a long transcript from the phage lambda promoter PL (Segawa and Imamoto, 1974; Franklin, 1974); but the relief of polarity is itself largely dependent on the lambda protein N. In a phage that joins the trp operon segment (trpD, C, B A) to a point distal to the N gene, lacking the tL site, synthesis of trp mRNA starting at the PL promoter continues even when translation is generally inhibited by chloramphenicol, but in the absence of functional N gene product synthesis of the mRNA can be blocked by the antibiotic. Unexpectedly, in the absence of N function, even when translation is occurring, weak termination of transcription occurs at some sites in the translocated trp operon.

Chloramphenicol↗
PubMed1978

PL-promoted transcription of the promoter-proximal N-trp region is insensitive to chloramphenicol in the absence of N function.

When the trp operon is translocated into the early region of lambda phage, transcription originated at the PL promotor is known to be modified by function of the N gene product so that transcription of the operon continues when translation is blocked by nonsense mutations or by ribosomal antibiotics. When N function is deficient in a phage that joins the trp operon to a point distal to the N gene, deleting the tL site, nonsense mutations (Franklin, 1974) or chloramphenicol (Nakamura et al., accompanying paper) again block transcription of the bacterial operon. However, here we report that transcription over about the first 800 nucleotide pairs starting from the PL promotor of the N-trp operon is still insensitive to chloramphenicol even in the absence of N function. The region covers the full N gene and the initial bit of the trp operon.

Chloramphenicol↗
PubMed1977

Function of the tof gene product in modifying chemical stability of trp messenger RNA synthesized from the PL promoter of lambda trp phage.

The trp operon translocated into the early region of phage lambda can be transcribed under the control of two promoters, the authentic trp promoter (Ptrptrp mRNA) and the PL promoter of the N gene (PLtrp mRNA) (Imamoto and Tani, 1972; Ihara and Imamoto, 1976a). PLtrp mRNA is stabilized with time after infection: at early times after infection chemical degradation of PLtrp mRNA is two-fold slower than for Ptrptrp mRNA, while at later times the stabilization of PLtrp mRNA is almost total. The stabilization of PLtrp mRNA is markedly reduced when the activity of the tof gene product is low due to a missense mutation of the tof gene. In contrast there is no significant reduction in stabilization when N function is lost by an amber mutation. On the basis of these and other experiments with lambdatrp susN7 tof12 phage, it is inferred that stabilization of the PLtrp mRNA is brought about by a modification of the "decay trigger", at least in part by the protein product of the tof gene.

Coliphages↗
PubMed1976

Effect of stringent and relaxed control on transcription of the tryptophan operon from the ptrp promoter and the PL promoter in trp phage.

Tryptophan (trp) mRNA synthesis from the authentic trp promoter (Ptrp) is apparently arrested upon translation blockage, while trp mRNA synthesis as a result of read-through from the Pl promoter of the N gene in trp phage is not so affected. When translation is blocked at a nonpermissive temperature in the temperature-sensitive mutants of Escherichia coli rel carrying altered ribosomal elongation factors G (strain CP78G) and Ts (strain HAK88), CP78G and HAK88 show relaxed and stringent phenotypes respectively in control of RNA synthesis. Under conditions causing translation blockage in both mutants, Pl-promoted synthesis of trp mRNA is not depressed while Ptrp-promoted synthesis of trp mRNA is blocked. The insensitivity of Pl-promoted transcription of the translocated trp operon to stringent control is also confirmed by using a strain 10b6s rel carrying a temperature-sensitive valyl-tRNA synthetase. In contrast to the above observation, transcription of the trp operon from either the Pl and Ptrp promoters in a 10b6r rel infected with trp is inhibited greatly at the nonpermissive temperature. This effect occurs even if the level of trp transcription observed at 30 degrees is already low, thus suggesting that translational machinery is intrinsically abnormal in the rel strain.

Coliphages↗
PubMed1976

In vitro transcription of the tryptophan operon in isolated bacterial nucleoids.

In vitro transcription of the trp operon in isolated nucleoids from Escherichia coli was studied. RNA synthesis in this system occurred primarily as a continuation of transcription which had been initiated in vivo; little or no initiation of new RNA chains was observed. Transcription of the trp operon in nucleoids by endogenous RNA polymerase procedded efficiently and ceases sequentially in the order of the gene sequence within the operon. Under these conditions, no appreciable exonuccleolytic digestion of nascent 3H-RNA was found, though some endonucleolytic cleavage was generally seen. Little or no incorporation of 14C-leucine into polypeptides was observed, inspite of tha fact that considerable number of ribosomes and nascent RNA chains were found attached to the isolated nucleoids. The synthesis of trp mRNA continued in the presence of chloramphenicol or fusidic acid, or under conditions where the rebosomal translocation factor G was inactivated. From these and other kinetic studies of trp mRNA synthesis in nucleoids obtained from nonsense strong polar mutants of the trp operon, it was shown that transcription in nucleoids was not connected functionally with transloational processes and thus unable to exhibit polarity effected by a nonsense mutation or by general translational blockage. In studies employing nucleoids from nonsense strong polar mutants of the trp operon, it was demonstrated that RNA polymerase are scantily distributed over the region downstream from the nonsense mutation site of the operon, thereby supporting a notion that in vivo transcription is eventually terminated near the nonsense mutation.

DNA, Bacterial↗
PubMed1976

Stability of "spacer" sequences of pre-ribosomal RNA in Escherichia coli.

"SPACER" SEQUENCES OF AN RRNA gene transcript were detected with high efficiency by hybridization with DNA of the specilized transducing phase phi80rrn. Hybridization-competition studies revealed that 20 to 23% of the 30S precursor rRNA, obtained from E. coli mutant strain AB301/105, consist of "spacer" sequences. The "spacer" sequences formed hybrids with E. coli DNA, but not with Vibrio DNA. Experiments with RNA labeling in the presence of rifampicin showed that more than 80% of the spacer sequences arrive in full-length 30S pre rRNA chains before any cleavage of the RNA occurs. The hybridization assays also permitted the detection of "spacer" sequences in pulse-labeled rRNA of wild-type cells, in which the 30S pre-rRNA is already cleaved during its synthesis. Many of these "spacer" sequences degraded to alcohol-soluble materials with a half-life time of 1.2 min. The half-life was not lengthened by the treatment of cells with chloramphenicol, which stabilizes bulk mRNA. However, unstable "spacer" sequences transcribed in cells deficient in RNase III exhibited slower degradation, with a half-life time of about 9 min, whereas the cleavage of 30S pre-rRNA to smaller RNA species occurred with a half-life of about 3 min. These results are consistent with the notion that a rate-limiting action of RNase III in the initial attack leads to degradation of "spacer" sequences in rRNA gene transcript; and that degradation is not at all connected with ribosome translocation.

Base Sequence↗