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F Blasi

Publications and source records attributed to F Blasi.

At least 235 records · Page 13Linked to original sources
PubMed1981

In vivo and in vitro detection of the leader RNA of the histidine operon of Escherichia coli K-12.

The DNA of the attenuator region of the histidine operon of Escherichia coli has been transcribed in a purified in vitro system and found to synthesize two major RNA transcripts. The first one, 180 nucleotides long, has been identified as the histidine-specific leader RNA. It contains the coding sequence for the leader peptide [Di Nocera, P. P., Blasi, F., Di Lauro, R., Frunzio, R. & Bruni, C. B. (1978) Proc. Natl. Acad. Sci. USA 75, 4276-4280] and is terminated at the attenuator site. Termination of transcription at this site is extremely efficient in the in vitro system. The leader RNA also has been detected in vivo in a minicell producer strain transformed with plasmids harboring the regulatory region of the histidine operon of E. coli. A second RNA molecule is synthesized in the in vitro system. It has a divergent direction of transcription with respect to the histidine leader RNA, but its role, if any, in the regulation of the histidine operon remains to be ascertained. The existence of the histidine leader RNA lends support to the regulatory mechanism which postulates that regulation of the histidine operon is dependent on the alternative secondary structures that the leader RNA may assume, depending on whether or not the histidine-rich leader peptide is translated.

Base Sequence↗
PubMed1980

Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors.

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.

Base Sequence↗
PubMed1978

Nucleotide sequence of the attenuator region of the histidine operon of Escherichia coli K-12.

The attenuator region of the histidine operon of Escherichia coli K-12 has a potential coding capacity for two peptides, one of 16 amino acids and another of 30 amino acids. This region is followed by a perfect palindrome of 14 base pairs separated by five nucleotides. A G+C-rich region precedes and follows a possible transcription termination sequence. These features are compatible with a model in which active translation of a leader mRNA interferes with transcription termination, thus causing derepression of the histidine operon. The sequence of the region coding for the hypothetical 16-amino acid peptide is of particular relevance because it indicates the site and a possible mechanism of action of histidyl-tRNAhis in regulating histidine gene expression. Seven contiguous histidine codons are present within this sequence: : formula: (see text)

Base Sequence↗
PubMed1977

Biochemical and regulatory properties of Escherichia coli K-12 hisT mutants.

Escherichia coli K-12 hisT mutants were isolated, and their properties were studied. These mutants are derepressed for the histidine operon, map close to the purF locus at about 49.5 min on the E. coli linkage map, and lack pseudouridylate synthetase activity. The defect in this enzyme leads to the absence of pseudouridines in the anticodon loop of several transfer ribonucleic acid species, as evidenced by the altered elution profile on reversed-phase chromatography and resistance to amino acid analogues. Finally, the hisT mutants studied have a reduced growth rate that appears to be linked to hisT, although it is not known whether it is due to the same mutation. The normal generation time can be restored by supplementing the medium with adenine, uracil, and isoleucine.

Adenine↗
PubMed1976

Gene expression of the histidine operon.

Histidine biosynthesis in Salmonella typhi-murium and Escherichia coli is regulated through repression and feedback inhibition. Repression is triggered by the intracellular level of histidyl-tRNA whereas feedback inhibition is brought about by histidine itself, an allosteric inhibitor of the first biosynthetic enzyme. Several lines of evidence indicate that these two processes are interconnected and the first biosynthetic enzyme is itself a regulatory molecule for the his operon. The regulation seems to be exerted in a negative fashion, although a positive control cannot be excluded.

ATP Phosphoribosyltransferase↗
PubMed1975

In vitro transcription of the Escherichia coli histidine operon primed by dinucleotides. Effect of the first histidine biosynthetic enzyme.

Initiation of transcription of the Escherichia coli histidine (his) operon in vitro has been analyzed. The DNA of a specialized transducing phage, ø80dhis, was used as a template, and his RNA was measured by RNA/DNA hybridization. Taking advantage of the fact that E. coli RNA polymerase cannot initiate transcription when the nucleoside triphosphates are present at very low (5 muM) concentration, his RNA initiation was primed by dinucleoside monophosphates. It has been found that his RNA synthesis can be stimulated by one of the three dinucleotides CpA, ApA, and ApG. Under these conditions, it is the initiation of his RNA synthesis that is stimulated. Stimulation of his RNA synthesis by the three dinucleotides apparently occurs at a single initiation site, as judged by the nonadditivity of the effects of the three dinucleotides. This was further confirmed by the effect of phosphoribosyltransferase (the first enzyme of histidine biosynthesis, which specifically represses the synthesis of his RNA) on ApA primed RNA synthesis. Addition of his protein results in a sharp decrease of his RNA synthesis, with no effect whatsoever on the levels of RNAa transcribed from other regions of the template. Our data suggest that the 5' -terminal sequence of his RNA made in vitro is ApApG and that the base immediately preceding this sequence is C.

Amidophosphoribosyltransferase↗
PubMed1975

Hamster alpha-amanitine-resistant RNA polymerase II able to transcribe polyoma virus genome in somatic cell hybrids.

A hamster cell line resistant to alpha-amanitine has been isolated (alpha-am-r, BHK-T6-G-1). Cell extracts of this mutant have an alpha-amanitine-resistant RNA polymerase II (nucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) activity as shown by DEAE-cellulose column chromatography. This mutation is dominant in interspecific hybrids with 3T3 mouse cells. In such hybrids polyoma virus can grow with equal efficiency in the presence or absence of the drug, thus indicating that the RNA polymerase of the unsusceptible parental cell can participate in the correct transcription of the viral genome.

Animals↗
PubMed1975

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

ATP Phosphoribosyltransferase↗
PubMed1973

Inhibition of transcription of the histidine operon in vitro by the first enzyme of the histidine pathway.

An in vitro system was developed for transcription of the histidine operon of Esherichia coli carried in the genome of a defective varphi80 transducing phage. The messenger RNA (mRNA) of the histidine operon synthesized in the in vitro system was detected by hybridization to single strands of both varphi80 and varphi80dhis DNA, and by competition of this hybridization with unlabeled histidine mRNA that had been synthesized in vivo (RNA extracted from cells in which the histidine operon had been derepressed). Under the conditions used, RNA complementary to the histidine operon was about 15% of the total RNA that was synthesized in vitro from the varphi80dhis DNA template. The RNA complementary to the histidine operon was synthesized on the "sense" strand (the R strand) of varphi80dhis in the form of a polycistronic message with a sedimentation coefficient (about 38 S) very close to that observed for the histidine mRNA synthesized in vivo. Synthesis of the histidine operon RNA appears to be subject to control in vitro. Addition of the first enzyme of the pathway for histidine biosynthesis blocked transcription of the histidine operon specifically, strongly suggesting that this enzyme acts as a regulatory protein for the histidine operon.

Cell-Free System↗
PubMed1973

Deletion mapping and orientation of the histidine operon of Escherichia coli on a transducing bacteriophage.

The defective prophage phi80ilambdac(I)857dhis has been mapped through both marker rescue and deletion analysis. Deletions have been isolated which put residual his genes close to trp genes. Analysis of these deletions shows that the histidine operon on the prophage is oriented clockwise as on the bacterial chromosome, thus opposite to the orientation of the trp operon. The presence of the his promoter-operator region is inferred by the ability of the prophage-carrying strain to derepress sequentially under conditions in which the histidine concentration is limiting. In addition to his, the gnd gene is also present on the prophage and is located between his and trp operons. The bacterial genes are inserted in the right arm of the prophage and substitute for all of the late function genes, except for the first three. These data indicate that the "sense" strand for transcription of the his operon in vivo must be the "R" strand.

Chromosome Mapping↗
PubMed1972

Isolation of transducing bacteriophages for the histidine and isoleucine-valine operons in Escherichia coli K-12.

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.

Centrifugation, Density Gradient↗
PubMed1971

Interaction between the first enzyme for histidine biosynthesis and histidyl transfer ribonucleic acid.

Previous studies suggested that phosphoribosyltransferase, which catalyzes the first step of the pathway for histidine biosynthesis in Salmonella typhimurium and which is sensitive to inhibition by histidine, plays a role in repression of the histidine operon. Recently, we showed that the enzyme has a high affinity for histidyl transfer ribonucleic acid (His-tRNA), which is known to participate in the repression process. In the present study, we have investigated further the interaction between the enzyme and His-tRNA. We found that His-tRNA binds at a site on phosphoribosyltransferase distinct from the catalytic site and the histidine-sensitive site; that the substrates of the enzyme inhibit the binding of His-tRNA, whereas histidine does not do so; that, once a complex has been formed between phosphoribosyltransferase and His-tRNA, the substrates of the enzyme decrease the stability of the complex, whereas histidine is without effect; and that purified phosphoribosyltransferase which has a defect in its inhibition by histidine (produced by mutation) displays an altered ability to bind His-tRNA, a finding which may be a reflection of the fact that mutants producing such a defective enzyme display an alteration of the repression process.

Acrylates↗
PubMed1970

Interaction between histidyl transfer ribonucleic acid and the first enzyme for histidine biosynthesis of Salmonella typhimurium.

Previous studies showed that the enzyme (phosphoribosyltransferase) which catalyzes the first step of the histidine pathway in Salmonella typhimurium plays a role in regulation of the histidine operon. Since histidyl transfer ribonucleic acid (His-tRNA) is required for repression of the histidine operon, we considered the possibility that the role of phosphoribosyltransferase might be realized through an interaction with His-tRNA. One prediction inherent in this idea is that the enzyme should interact with His-tRNA in vitro. Evidence is presented for such an interaction. Binding of (3)H-His-tRNA to purified phosphoribosyltransferase was tested on Sephadex columns and on nitrocellulose filters. The enzyme was found to have a high affinity for tRNA. Comparing the binding of (3)H-His-tRNA with that of tRNA aminoacylated with other (3)H-amino acids disclosed that the binding of the histidyl species of tRNA is favored over that of other species and is dependent upon magnesium-ion concentration.

Acrylates↗