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PubMed1991

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803.

The two operons atp1 and atp2, encoding the subunits of the F0F1 ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F0F1 ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5' end of atp1, coding for a putative gene product similar to uncI in Escherichia coli. A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.

Amino Acid Sequence↗

PubMed1976

Initial trp operon sequence in Escherichia coli is transcribed without coupling to translation.

The 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 (trp OAEG) retaining only 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 trp OAE14. 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 trpOAE14 was 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.

DNA, Bacterial↗

PubMed1976

A transcriptional barrier in the regulatory region of the tryptophan operon of Escherichia coli: its role in the regulation of repressor-independent RNA synthesis.

A study was made of the influence of the growth rate of Escherichia coli bacteria on the transcription of the tryptophan (trp) operon, in various trp repressor negative strains. Selective measurement of the levels of RNA transcribed from the regulatory region (reg) of this operon and from the structural genes, respectively, has revealed that the increase of the rate of trpRNA synthesis with bacterial growth rate (Rose and Yanofsky, 1972) is due to a decrease of the frequency of termination of transcription at the transcriptional barrier in the regulatory region of the operon. In a mutant strain of E. coli with a deletion covering the promotor distal part of the regulatory region of the trp operon where the barrier is located, the rate of trpRNA synthesis is not affected by the growth rate. In suA- strains, in which Rho factor activity is reduced the bacterial growth rate does not affect the rate of synthesis of trpRNA. This result suggests that in wild type bacteria Rho factor contributes to the control of the transcription of the trp operon. In bacteria with a mutation rendering Tryptophanyl-tRNA synthetase (TRSase) inactive (trpS- strains) the rate of trpRNA synthesis is affected by the growth rate in the same way as in the isogenic wild type bacteria. This result indicates that TRSase plays no obligatory role in the control of trpRNA synthesis through a mechanism of termination and anti-termination of transcription, at least not in the studied strains, which carried a relA mutation.

Binding, Competitive↗

PubMed1987

Promoter switching during development and the termination site of the sigma 43 operon of Bacillus subtilis.

Sequencing data indicated that the RNA polymerase sigma 43 operon of Bacillus subtilis consisted of three genes, P23 (function unknown), dnaE (DNA primase), and rpoD (sigma 43) (Wang and Doi 1986a). S1 nuclease mapping experiments with RNA from various stages of growth demonstrated the presence of two overlapping sigma 43 promoters that controlled the expression of the operon during growth and a sigma 37 promoter that regulated the expression of the operon during the sporulation phase. This promoter switching mechanism ensured that this important operon would be expressed during different nutritional states of the cell and also illustrated a function for the minor RNA polymerase sigma 37 holoenzyme in the expression of genes which are normally expressed during the logarithmic phase of growth. The location of the transcription termination signal confirmed that the sigma 43 operon consists of three genes.

Bacterial Proteins↗

PubMed1986

Analysis of transcription and processing signals of the 16S-23S rRNA operon of Mycoplasma hyopneumoniae.

The 16S and 23S rRNA genes of Mycoplasma hyopneumoniae are closely spaced in one operon. The two genes are separated by a spacer region of 500 bp which shows no sequence homology to bacterial tRNA genes. Within this operon seven 5' and five 3' ends of various rRNA species were mapped and the corresponding DNA was sequenced. The results are consistent with the following model for synthesis of rRNAs: Transcription of the operon is initiated from either of two tandemly arranged promoters leading to a large precursor RNA consisting of both 16S and 23S rRNAs. This primary transcript is first cleaved within stem structures surrounding the two rRNAs to yield premature 16S and 23S rRNAs. By further processing events the mature 5' and 3' ends are generated. The promoter sequences of this operon differ from those of other eubacterial promoters in lacking the typical -35 region. The putative termination site at the 3' end of the operon is reminiscent of rho-independent terminators in Escherichia coli.

Base Sequence↗

PubMed1988

Cloning and transcription analysis of the entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) and identification of a closely associated transcription unit.

The entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) was cloned and its transcriptional organization and regulation was analyzed by Northern blotting, S1 nuclease mapping and transcriptional fusions. Transcription of the operon is glycerol-inducible and glucose-repressible; glyA (presumptively encoding glycerol kinase), gylB (encoding sn-glycerol-3-phosphate dehydrogenase) and gylX (a non-essential 1.1 kb sequence) are transcribed consecutively to give a 5.4 kb mRNA. Two alternative transcription termination or gyl mRNA processing sites are located within the operon; one (a discrete site) lies between gylB and gylX and the other (a heterogeneous site) positioned 3 kb into the operon, may correspond to the gylA-gylB intercistronic region. A 0.9 kb glycerol-inducible transcription unit is located immediately upstream of gylABX. Transcriptional fusion studies employing an attP site-deleted phage vector provided complementary evidence for the organization of the operon.

Chromosome Mapping↗

PubMed1982

Influence of the rho-15 temperature-sensitive (ts) mutation on the expression of the deo-operon in Escherichia coli.

In the rho-15 temperature-sensitive (ts) mutant deo-operon enzymes show no sensitivity to catabolite repression and are not derepressed under the influence of a constitutive regulatory mutation, cytR. These data suggest that intact Rho-protein along with CRP protein is necessary for a catabolite sensitive deo-operon promoter cytP to work. In addition, there are data suggesting that Rho-factor and CRP-protein interact with each other in regulation of the deo-operon. Thus, in studies of the effect of the rho-15 (ts) and crp mutations, maximum deo-enzyme levels have been found in the double rho-15 (ts) crp mutant, and therefore intact Rho-protein in the crp genome or intact CRP-protein on the rho-15 (ts) background seems to be an obstacle for the deoP promoter in the deo-operon. In rho-15 (ts) a relative increase has been observed in the enzyme activity for a distal purine nucleoside phosphorylase gene with respect to a proximal thymidine phosphorylase gene. However in crp, the rho-15 (ts) mutation has no effect on the polarity gradient, that is on the background of impaired CRP protein Rho-factor does not seem to work as a transcription terminator within the operon.

Escherichia coli↗

PubMed1995

The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus.

The synthesis of virulence factors and other exoproteins in Staphylococcus aureus is controlled by the global regulator, agr. Expression of secreted proteins is up-regulated in the postexponential growth phase, whereas expression of surface proteins is down-regulated by agr. The agr locus consists of two divergent operons, transcribed from neighboring but non-overlapping promoters, P2 and P3. The P2 operon sequence, reported here, contains 4 open reading frames, agrA, C, D, and B, of which A and C appear to encode proteins of a classical 2-component signal transduction pathway. The P3 operon specifies a 0.5 kb transcript, RNA III, which is the actual effector of the agr response, and, incidentally, encodes the agr-regulated peptide delta-hemolysin. Transcriptional fusions have shown that both P2 and P3 are agr sensitive (function in an agr+ but not in an agr- background) and deletion analysis has shown that all four of the P2 ORFs are involved; agrA and agrC seem to be absolutely required for the transcriptional activation of the agr locus, whereas agrB and agrD seem to be partially required. Since transcription of P2 requires P2 operon products, the P2 operon is autocatalytic, and is thus admirably suited to the need for rapid production of exoproteins at a time when overall growth is coming to a halt.

Amino Acid Sequence↗

PubMed2001

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria.

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Amino Acid Sequence↗

PubMed2005

A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl-CoA in Methanosarcina thermophila.

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates.

Acetyl Coenzyme A↗

PubMed1996

Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60.

The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a -35/-10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45-75 min after shifting from 28 degrees C to 39 degrees C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.

Adenosine Triphosphatases↗

PubMed2002

Analysis of the cellular functions of Escherichia coli operons and their conservation in Bacillus subtilis.

The common assumption of operons as composed of genes that cooperate in a biological process is confirmed here by showing that Escherichia coli operons tend to be composed of genes that belong to the same general class of cellular function. Furthermore, the comparison between the genomic organization of E. coli and that of Bacillus subtilis shows that the genes that are homologous to genes that belong to experimentally characterized E. coli operons tend to cluster in neighboring regions of the genome. This tendency is greater for the subset of E. coli operons whose genes belong to a single functional class. These observations indicate strong evolutionary pressure that, translated into functional constraints, leads to the inclusion of many essential functions in conserved operons and clusters in these two distant species.

Bacillus subtilis↗

PubMed2005

Post-transcriptional operons and regulons co-ordinating gene expression.

Experiments reported over the past several years, including genome-wide microarray approaches, have demonstrated that many eukaryotic RNA-binding proteins (RBPs) associate with multiple messenger RNAs (mRNAs) both in vitro and in vivo. This multi-targeted binding property of RBPs has led to a model of regulated gene expression in eukaryotes that we termed the post-transcriptional operon. This concept was established by an analogy between polycistronic mRNAs that are generated from bacterial operons, and the co-ordinated regulation of multiple monocistronic mRNAs by RBPs. Post-transcriptional operons represent a powerful mechanism to organize and express genetic information as functionally related combinations of monocistronic mRNAs. In fact, much of the diversification of individual proteomes may be determined by the combinatorial properties of post-transcriptional operons. This review examines data supporting the role of post-transcriptional operons and regulons in organizing genetic information and co-ordinating expression of functionally related transcripts from their origins at transcription to their subsequent splicing, export and translation.

Bacteria↗

PubMed1983

Transcription attenuation is the major mechanism by which the leu operon of Salmonella typhimurium is controlled.

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Bacteriophage lambda↗

PubMed1984

Point mutations that reduce the expression of malPQ, a positively controlled operon of Escherichia coli.

malPQ is one of three operons controlled by the positive regulator gene malT. With the objective of defining DNA sequences essential for malPQ transcription, we looked for cis-dominant mutations that reduced the level of expression of this operon. We first constructed malP-lac fusion strains, selected from one of them a series of mutants resistant to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease), and retained the clones that contained a mutation reducing the expression of the hybrid operon in a cis-dominant fashion. Nineteen such mutations were sequenced, and their effect on an otherwise wild type malPQ operon was studied. Three of them mapped in a transcribed portion of the operon, and are believed to exert their effect at the translation level. The others map upstream from the transcription startpoint (co-ordinate +1) and help define three DNA segments that must play a predominant role in transcription initiation: the Pribnow box (from positions -7 to -12); and two inverted repeats, extending from position -32 to -36, and -59 to -63, respectively, which are proposed to constitute part of the binding site for MalT protein.

Base Sequence↗

PubMed1984

Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12.

A 4509 base-pair DNA fragment containing the phenylalanine and tyrosine operons of Escherichia coli K12 has been sequenced, and the pattern of transcription of these operons examined by S1 mapping, primer extension and galK fusion analyses. The phe operon consists of promoter, operator, leader region containing the phe attenuator and the pheA gene encoding chorismate mutase/prephenate dehydratase. The tyr operon consists of promoter, operator, a short leader region without an attenuator, and two structural genes aroF and tyrA encoding the tyrosine-sensitive isoenzyme of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase and chorismate mutase/prephenate dehydrogenase, respectively. A bidirectional transcription terminator occurs between the two operons. The predicted amino acid sequences of chorismate mutase/prephenate dehydrogenase and chorismate mutase/prephenate dehydratase are homologous at their N termini, while the tyrosine-sensitive isoenzyme of DAHP synthetase is closely homologous to the phenylalanine-sensitive isoenzyme encoded by aroG.

Amino Acid Sequence↗

PubMed1987

High-affinity L-arabinose transport operon. Gene product expression and mRNAs.

Various portions of the "high-affinity" L-arabinose transport operon were cloned into the plasmid expression vector pKK223-3 and the operon-encoded protein products were identified. The results indicate that three proteins are encoded by this operon. The first is a 33,000 Mr protein that is the product of the promoter-proximal L-arabinose binding protein coding sequence, araF. A 52,000 Mr protein is encoded by sequence 3' to araF and has been assigned to the araG locus. The sequence 3' to araG encodes a 31,000 Mr protein that has been assigned to the araH locus. Both the araG and araH gene products are localized in the membrane fraction of the cell, implying a role in the membrane-associated complex of the high-affinity L-arabinose transport system. Nuclease S1 protection studies indicate that two operon message populations are present in the cell, a full-length operon transcript and a seven- to tenfold more abundant binding protein-specific message. The relative abundance of these two message populations correlates with the differential expression of the binding protein and the membrane-associated proteins of the transport system.

Arabinose↗

PubMed1987

Regulation of alpha operon gene expression in Escherichia coli. A novel form of translational coupling.

The alpha operon of Escherichia coli contains the genes for ribosomal proteins S13, S11, S4, RNA polymerase subunit alpha, and r-protein L17, in this order. Previous studies have shown that translation of all four ribosomal proteins is regulated by S4, and that binding of S4 to the mRNA at the start site for S13 translation is probably responsible for the regulation of translation of S13, S11 and S4. The alpha gene is "unique" in that it is located between the genes for two ribosomal proteins (S4 and L17) and yet appears to be regulated independently of them. In the present studies, we have measured the synthesis rates of all the alpha operon proteins under a variety of physiological conditions. Our results confirm that alpha gene expression is regulated independently of the co-transcribed ribosomal protein genes and is relatively insensitive to translational feedback repression by S4. S1 nuclease analysis of alpha operon mRNA failed to reveal the presence of any unique transcription start or mRNA cleavage that leads to separation of the alpha cistron from preceding ribosomal protein cistrons. Therefore, it appears that differential regulation of alpha synthesis takes place at the level of mRNA translation. We have also carried out a deletion analysis of the alpha operon leader and identified a region of the alpha operon leader mRNA that is required for regulation by S4. Furthermore, deletion of this region results in increased synthesis of L17 together with S13, S11 and S4, whereas alpha synthesis did not increase significantly. Therefore, we conclude that interaction of S4 with this single target site results in translational repression of not only the proximal three cistrons for S13, S11 and S4 but also that of the last cistron, L17, without affecting the intervening alpha cistron.

Escherichia coli↗