Yasunobu Kano

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.

Decay of individual Escherichia coli trp messenger RNA molecules is sequentially ordered

Two new features of trp mRNA decay were observed by following the metabolism of chains specifically labeled in their 3′-terminal A -gene sequences. Further formation of RNA was shut down with rifampicin and tryptophan, and the decay and size distribution of chains containing labeled A segments were assessed. The 3′-terminal A gene sequences decayed only after a lag of several minutes. During the lag, intermediates in decay could be extracted from cells. They sedimented in sucrose gradients at positions expected for trp operon mRNA lacking successive gene equivalents from the 5′-end. These results eliminate models for mRNA decay in which molecules begin to decay in bulk from their 3′-ends. Quantitative analyses also eliminate a model in which each gene sequence is at jeopardy from the time of its synthesis. Instead the results are consistent with decay proceeding through a mRNA from a random start at or near the 5′-end, with hold-up points at each inter-cistronic border. When distal portions of long species of total Escherichia coli mRNA other than trp were labeled, there was again a lag of several minutes before the labeled sequences broke down to acid-soluble fragments. During the lag, the labeled segments could again be extracted in progressively shorter chains. In principle, similar analyses can determine the mode of decay of other polycistronic mRNA species.

Isolation of the novel regulatory mutants of the tryptophan biosynthetic system in Escherichia coli

SummaryA novel type of tryptophan requiring mutants of Escherichia coli was isolated. The mutation maps between str and malA.These mutants, designated as trpS, have alterations in the regulation of the tryptophan operon. Neither derepression nor complete repression of the tryptophan biosynthetic enzymes was observed with this mutant. Dominance test shows that the trpS mutation is recessive to the wild type allele. TrpS mutant, therefore, is a type of super-repressed mutants distinct from is mutant in the lactose system of E. coli.It was found that the tryptophanyl-tRNA synthetase is specified by the trpS gene. This indicates that the transfer mechanism of tryptophan is related to repression of the tryptophan operon.

Initial trp Operon Sequence in Escherichia coli is Transcribed without Coupling to Translation

SummaryThe transcription of the “leader” region (Bronson et al., 1973) of the trp operon in Escherichia coli was studied in normal mutants which delete most of the operator-distal region of the operon [a deletion strain (trpOAE6) retaining onoy about one third of the “leader” region and two deletion strains (trpOAE14 and trpOAE2) retaining the whole “leader” region and an initial portion of the trpE], as well as in a strain with an intact trp operon, but with a temperature-sensitive lesion in ribosomal protein factor EFTs (strain HAK88).In these deletion mutants, mRNA molecules corresponding to the “leader” region were detected as most of the trp-specific mRNA. Less inhibition of transcription, of the promoter-proximal portion of the trp“leader” region than that of more distal genes of the operon, was found in chloramphenicol-treated cells of strain trpOAE14. It was also observed that transcription of the initial one third portion of the “leader” region was not repressed by tryptophan in strains trpOAE6 and trpOAE14. A similar effect of a translation block on transcription of the distal part of the “leader” region was observed with strain HAK88 at the nonpermissive temperature.In sedimentation analysis of polyribosomes containing the trp mRNA molecules from the deletion mutants, trp mRNA from strain trpOAE14was found in monosomes and small polyribosomes, whereas the majority of the trp mRNA from strain trpOAE6 was found joined to a single ribosome or ribosomal subunit.These results suggest that ribosomes bind in vivo to a site(s) located in the middle of the “leader” mRNA sequence, and that the initial transcription of the trp operon does not require any connection to functional translational machinery, while continuation of RNA synthesis beyond a first ribosome binding site seems indispensably coupled to ribosome function.

Involvement of the nusA and nusB gene products in transcription of Escherichia coli tryptophan operon in vitro

SummarySynthesis of trp mRNA in vitro directed by plasmid DNA carrying the entire trp operon was studied using crude protein extracts (S-100) from Escherichia coli strains carrying the nusA or nusB mutation or both. It was found that the levels of trp mRNA transcribed from the promoter-distal genes (trpCBA) relative to that from the promoter-proximal genes (trpED) was markedly lower with extracts from the nus- mutants than that from the nus+ strain. Kinetic experiments suggest that termination of RNA transcripts at intragenic transcriptional barriers is prevented by the nus gene products from allowing efficient experession of the operon.

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

SummaryThe 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.

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

SummaryWhen 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 λ promoter PL(Segawa and Imamoto, 1974; Franklin, 1974); but the relief of polarity is itself largely dependent on the λ 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 tLsite, synthesis of trp mRNA starting at the PLpromoter 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.

Comparative studies of the effect of DNA superhelicity on in vitro transcription catalyzed by Escherichia coli S100 proteins and purified RNA polymerase

The effect of DNA superhelicity on in vitro transcription catalyzed by purified Escherichia coli RNA polymerase or S100 crude extract proteins was examined at various KCl concentrations. DNA from a recombinant plasmid pMT48 harboring the pL promotor-controlled fused N-trp genes and the pR promotor-controlled tof (cro) gene was employed as a template. Stimulation of transcription by superhelicity is generally more pronounced with the S100 crude extract proteins than with pure RNA polymerase. At KCl concentrations lower than 100 mM with pure RNA polymerase, there is no significant difference in the template activity between the supercoiled and relaxed forms of pMT48 DNA. In contrast, the dependence of efficient template activity on superhelicity is great over a whole range of KCl concentrations from 1.7 to 400 mM in the system using the S100 crude extract. The relative insensitivity of the pR promotor to superhelicity can be observed in either transcription assay system. Analysis of the kinetics of pL-promoted synthesis of trp mRNA indicates that diminished transcription in vitro on a relaxed template results mainly from less frequent RNA chain initiations, but at least in part from premature arrest of the chain elongation.

Stability of “spacer” sequences of pre-ribosomal RNA inEscherichia coli

Summary“Spacer” sequences of an rRNA gene transcript were detected with high efficiency by hybridization with DNA of the specialized transducing phage ϕ80rrn. Hybridization-competition studies revealed that 20 to 23% of the 30S precursor rRNA, obtained formE. coli mutant strainAB301/105, consist of “spacer” sequences. The “spacer” sequences formed hybrids withE. coli DNA, but not withVibrio 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 wildtype 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.

Translation-uncoupled transcription of promoter-proximal DNA sequences in E. coli strains harboring mutationally-generated constitutive promoters within genes of the trp operon.

SummaryBy an extension of previously described methods (McPartland and Somerville, 1976) we have isolated several prototrophic mutants of Escherichia coli harboring strong constitutive promoters which lie within the trp operon. The production of trp mRNA from two such groups of mutants has been studied in detail. For mutants whose promoter sites lie near the operator-distal end of trpD, mRNA corresponding to the trpCBA genes is transcribed constitutively at 40–53% of the rate characteristic of high level transcription from trpP1. Strains with promoters within trpC are capable of constitutive trpCBA mRNA production at 19–24% of the maximal trpP1 rates. Under conditions of tryptophan repression, these strains transcribe little or no mRNA from the region of trp DNA between trpP1 and the internal promoters.When translation was blocked by chloramphenicol, the constitutive synthesis of trp mRNA corresponding to the distal trp genes was abolished, while synthesis of a small initial trp mRNA fragment continued. Like transcription of the initial bit of the normal trp operon (Kano et al., 1976), transcription beyond the initiation site for polypeptide synthesis of the first intact structural gene is coupled to translation, but transcription prior to the initiation site is not. We suggest that the first ribosome binding site —whether in a “leader”-like sequence or in a structural gene—is the likely place where chloramphenicol exerts its effect and that the continuation of transcription is then blocked by the arrested ribosome.