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PubMed1983

Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli.

The L11 ribosomal protein operon in Escherichia coli consists of the structural genes for proteins L11 and L1. Hybrid deletion plasmids were constructed carrying these two genes with decreasing amounts of the leader mRNA under lac transcriptional control. Measurements of mRNA and protein synthesis directed by these plasmids in vitro and in vivo demonstrate that the regulation of this operon is posttranscriptional and identifies a region of the mRNA, preceding the proximal L11 gene, important for successful feedback inhibition of L11 and L1 synthesis by L1. Additionally, deletions extending to the ribosome binding site of the L11 gene fail to synthesize both L11 and the downstream L1 protein although synthesis of the corresponding mRNA remains unchanged. These results directly demonstrate the presence of translational coupling in this bicistronic operon.

Bacterial Proteins↗

PubMed1986

The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease.

Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant nuclear genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We report here that the virD operon encodes a site-specific endonuclease that cleaves at a unique site within each of the 24 bp direct repeats that flank the T-DNA. The endonuclease function was further localized to the 5' end of this operon by demonstrating that cleavage does not occur in virD mutant strains of Agrobacterium and that the 5' end of the virD operon is sufficient to direct cleavage in E. coli. Analysis of nucleotide sequence and protein data indicate that two proteins of 16.2 and 47.4 kd are involved.

Amino Acid Sequence↗

PubMed1987

Differential mRNA stability controls relative gene expression within a polycistronic operon.

In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon. The polycistronic malEFG operon of E. coli contains two REP sequences (highly conserved inverted repeats) within the malE-malF intercistronic region. Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein. A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA. The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common.

ATP-Binding Cassette Transporters↗

PubMed1993

Analysis of the mRNA structure of the Pseudomonas putida TOL meta fission pathway operon around the transcription initiation point, the xylTE and the xylFJ regions.

The 13 genes encoded by the meta-cleavage operon (approx. 11 kb) of Pseudomonas putida TOL plasmid pWW0 are transcribed from a single promoter, Pm. In P. putida, transcription from Pm was strictly dependent on the presence of effector-activated XylS protein. Three regions of the transcript were analyzed in the wild-type strain of P. putida (pWW0) by S1 nuclease protection and primer extension analyses. A major point of transcription initiation was found in the most 5'-end of the operon, which defined a -10/-35 promoter. Another main mRNA 5'-end point located within the coding sequence of the first gene of the operon was detected and identified as a stable endonucleolytic cleavage product, which rendered this gene inactive for further translation. The estimated half-life of this leader region was 2 min. Analyses of the transcripts at xylLTE and xylFJ regions revealed the presence of several relatively stable mRNA products with 5'-ends located along these regions, which arose from specific endonucleolytic mRNA cleavage. The degradation of the stable intermediate products observed after the addition of rifampicin was similar for all the products, presenting a delay of approx. 10 min, followed by an exponential decay, with a half-life that ranged between 6 and 10 min.

Amino Acid Sequence↗

PubMed1995

Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli.

The sequence of the gat operon for galactitol (Gat) utilization from a wild-type isolate of Escherichia coli, strain EC3132, is presented. The operon comprises 7 open reading frames (ORFs) called gatYZABCDR. The genes are transcribed from a promoter located upstream of gatY. Genes gatABC encode the substrate-specific domains IIA, IIB and IIC of a galactitol-specific Enzyme II (EIIGat) of the phospho enol pyruvate-dependent carbohydrate:phosphotransferase system (PTS); gatD encodes an NAD-dependent Gat 1-phosphate dehydrogenase; and gatY an enzyme which hydrolyses tagatose 1,6-bisphosphate; gene gatZ is required in a cell to show a Gat+ phenotype, but its physiological function has not yet been identified; gatR encodes a repressor for the gat operon. All genes are highly similar to the gat genes from E. coli K-12; in this organism they map at 46.70 min of the gene map, equivalent to about 2180-2186 kbp.

Amino Acid Sequence↗

PubMed1994

Sequence of the sor-operon for L-sorbose utilization from Klebsiella pneumoniae KAY2026.

We have sequenced the complete sor-operon of Klebsiella pneumoniae KAY2026. The operon has been mapped at 91 min on the Klebsiella gene-map. It comprises seven open reading frames for the genes sorCDFBAME, which are expressed from the single promotor sorCP. The gene sorC codes for a regulator protein that positively and negatively regulates the expression of the operon; sorD encodes a D-glucitol-6-phosphate dehydrogenase, the genes sorFBAM encode four proteins of a phosphoenolpyruvate-dependent L-sorbose-phosphotransferase system and sorE, finally, an L-sorbose-1-phosphate-reductase.

Amino Acid Sequence↗

PubMed1991

Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis.

An insect larval toxin designated CryII is produced by several subspecies of Bacillus thuringiensis and differs from the other major delta-endotoxins in these bacteria in its size, toxicity profile and presence as part of an operon with three open reading frames (ORF). Such an operon from a novel B. thuringiensis isolate has been cloned and differs from one previously characterized in the following ways: (a) the size and number of amino acid repeats in one of the ORFs; (b) the smaller size of the CryII protoxin and the presence of a unique 110-kDa CryII-related antigen; and (c) high larvicidal activity for a particular Lepidopteran but low activity for a Dipteran. Various subclones of this operon were introduced into a plasmid-free B. thuringiensis strain and only the cryII gene was found to be necessary for protoxin accumulation.

Amino Acid Sequence↗

PubMed1980

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene.

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.

Bacteriophage lambda↗

PubMedDate not supplied

Sequence and properties of operator mutations in the bio operon of Escherichia coli.

The nucleotide-sequence changes occurring in newly isolated operator-constitutive mutations of the divergently transcribed bio operon have been determined. The observed point mutations are single GC leads to AT changes which occur at two symmetrical points in the hyphenated inverted repeat present in the control region. The changes at position -15 (with respect to the center of the inverted repeat) cause constitutivity of leftward operon expression and decreased expression of bioB, due to alteration of the -35 region of the rightward promoter. The change at position +15 is identical to one of the changes that Otsuka and Abelson (1978) detected in the bio p98 bio o34 double mutant. The location of the bio p131::IS1 insertion, which affects both leftward and rightward transcription, is also within the operator region. Both of the operator-constitutive mutations and the bio p131 insertion cause decreased repressor binding in vivo as shown by repressor titration tests on multicopy plasmids which bear them. The operator-constitutive mutations also decrease repressor binding in vitro, where added repressor fails to protect the TaqI site that is protected by repressor in bio o+ DNA. These results confirm several aspects of the model for bio operon regulation proposed by Otsuka and Abelson (1978).

Bacteriophage lambda↗

PubMed1983

Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli.

In Escherichia coli, radiation and chemically inducible mutagenesis requires a functional umuC gene product. The umuC mutants are defective in mutagenesis and slightly sensitive to DNA damaging agents. A chromosomal fragment that complemented the umuC mutations for UV mutability and UV resistance was cloned into miniF vector plasmid pMF3 by a shotgun method. A restriction map of the hybrid plasmid was constructed. Further subcloning, Tn1000 insertion inactivation, and complementation tests revealed that there are two genes, umuD and umuC in the former umuC region. The gene products of umuD and umuC were identified by the maxicell method to be proteins with Mr of 18 000 and 46 000, respectively. The two genes comprise an operon, and the transcriptional direction is from umuD to umuC. A plasmid carrying an umuC'-lac'Z gene fusion was constructed in vitro to study the regulation of the umu operon. It was shown that the umu operon is inducible by UV and chemical mutagens, and is regulated by the recA and lexA genes.

Bacterial Proteins↗

PubMed1984

Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida.

The transcription initiation site of the xylDEGF operon on the TOL plasmid of Pseudomonas putida mt-2 was determined in P. putida and in Escherichia coli by S1 nuclease and reverse transcriptase mapping. The induced synthesis of mRNA started at the same start point in both P. putida and E. coli, although the amount of mRNA in E. coli cells was less than that in P. putida. The nucleotide sequence of the region surrounding the start point was also determined. The ribosome-binding site (RBS) complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli preceded the predicted start codon for the xylD gene. The consensus nucleotide sequence for E. coli promoters was not found in the region preceding the transcription start point. On the other hand, the sequences of the "-10" and the "-35" regions of the xylDEGF operon revealed some homology with the respective, previously determined sequences of the xylABC operon of the TOL plasmid.

Base Sequence↗

PubMed1984

Studies on deo operon regulation in Escherichia coli: cloning and expression of the deoR structural gene.

Recombinant plasmid pBR322 derivatives containing the Escherichia coli deoR structural gene (coding for one repressor of the deo operon) and a mutant allele of the cmlA gene (chromosomally encoded chloramphenicol resistance) have been constructed and the positions of these genes on a 6.3-kb EcoRI fragment have been determined. Transformation of an E. coli deoR single mutant with any of the deoR+ plasmids resulted in complementation of the chromosomal deoR mutation. More importantly, however, transformation of a deoR cytR double mutant with the deoR+ plasmids also resulted in complete repression of Deo enzyme synthesis. Based on these data, we conclude that transcription of the deo operon initiating from both the cAMP/CRP-independent promoter-operator site, PO1, and the cAMP/CRP-dependent promoter-operator site, PO2, is negatively controlled by the deoR-encoded repressor, whereas the cytR-encoded repressor regulates deo operon expression only from the cAMP/CRP-dependent promoter-operator site, PO2.

Bacterial Proteins↗

PubMed1985

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB.

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.

Bacillus subtilis↗

PubMed1986

The organization of the araBAD operon of Escherichia coli.

The nucleotide (nt) sequence of the araBAD operon of Escherichia coli B/r has been determined. The nt sequence predicts a transcript of about 4250 nt. The coding regions of araB, araA and araD genes were identified by partial amino acid (aa) sequences of the purified proteins and their cleavage products. We have deduced that the polypeptides encoded by the araBAD operon consist of 566 (araB), 500 (araA) and 231 (araD) aa residues. The operon contains a leader sequence of 27 nt. The intergenic region between araB and araA is 10 bp in length, while the araA and araD genes are separated by 283 bp consisting primarily of six REP sequences. Comparison of the E. coli sequence with that of Salmonella typhimurium [Lin et al., Gene 34 (1985) 111-122; 123-128; 129-134] shows that, while considerable divergence in nt sequence has occurred, the aa sequences are largely conserved. The most pronounced difference, found in the araD gene, is the deletion of a single nt in the S. typhimurium sequence.

Amino Acid Sequence↗

PubMed1986

Heterologous expression in Bacillus subtilis. II. In vitro removal of the attenuator sequence of the Escherichia coli his operon allows expression of the cloned hisG gene in B. subtilis.

The promoter-proximal region of the Escherichia coli histidine (his) operon, including the promoter, the attenuator and the hisG gene, as well as the first of the nine structural genes of the his operon, have been cloned in Bacillus subtilis. In this host, the hisG gene could not be expressed because its transcription appeared to be irreversibly terminated at the attenuator (Ferretti et al., 1984). When the attenuator plus various lengths of the two bordering regions were removed, one of the attenuatorless sequences cloned in B. subtilis allowed the progression of transcription and complementation of the corresponding hisA mutation in this Gram-positive host. The deletion removed a 349-bp segment which contained the his attenuator and promoter. In B. subtilis, the productive transcription of the hisG gene started at a site in pAT153 and terminated in pC194. Sequence analysis of the deletion indicates that the E. coli ribosome-binding site of the his operon was used for the translation of the E. coli hisG gene mRNA in B. subtilis cells, which can thus grow in the absence of histidine.

Bacillus subtilis↗

PubMed1988

Transcriptional regulation and gene arrangement of Escherichia coli, Citrobacter freundii and Salmonella typhimurium biotin operons.

The bio operons of Citrobacter freundii and Escherichia coli K-12 (strain C600) were isolated by screening lambda banks for complementation of E. coli bio mutants. These were compared with the previously isolated bio operon of Salmonella typhimurium and previous data on E. coli K-12. The restriction maps of the operon are very different in the three species, but no difference in gene order was found. Operator-promoter DNA, identified by repressible titration and by biotin-repressible transcription in E. coli, was sequenced and compared to the published E. coli K-12 sequence. In the segment previously identified as operator/bioB promoter, C. freundii and S. typhimurium DNA are identical and differ from E. coli only by 2 bp. The DNA to the right of this segment (indicated by previous data to be the bioA promoter of E. coli) has diverged in all three species, and only E. coli has a sequence resembling a consensus promoter.

Bacteriophage lambda↗

PubMed1988

The primary structure of the operons coding for Shigella dysenteriae toxin and temperate phage H30 shiga-like toxin.

Nucleotide(nt) sequences were determined for the toxin (SHT) operon present in the chromosome of Shigella dysenteriae 1 and for the shiga-like toxin (SLT) operon found in the lambdoid phage H30 genome. The coding sequences of the sht and slt genes differ in 4 nt with 1 nt change responsible for an amino acid replacement. The deduced amino acid sequence in the A chain of the toxins is highly homologous to that of the A chain of ricin, a plant toxin. SHT-coding mRNAs were detected by mapping the 5' termini and using blot-hybridisation; one of them was more abundant and coded only for the B subunit of SHT while the other (bi-cistronic mRNA) encoded both subunits. An IS element related to the IS3 element of Escherichia coli was found in the chromosome of S. dysenteriae near the sht operon.

Amino Acid Sequence↗

PubMed1990

Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon.

A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics. Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class. In these strains the total cAMP synthesis was significantly lower than in a strain deleted only for the crr gene. This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme IIIGlc, is involved in adenylate cyclase (AC) activation or cAMP excretion. It was further shown that deletions in the pts operon do not affect synthesis of AC.

Adenylyl Cyclases↗