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PubMed1991

Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa.

The nitrite reductase gene (denA) and the cytochrome c-551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome. We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.

Anaerobiosis↗

PubMed1993

A putative transcription factor binding to the upstream region of the puf operon in Rhodobacter sphaeroides.

Gel shift assays of the upstream region of the puf operon in Rhodobacter sphaeroides were performed using cell-free extracts from cells grown under various culture conditions. The results suggested that a protein binding to the upstream region functioned as a repressor-like substance of the expression of the operon by oxygen tension or light. The density of the shifted band of cell-free extracts from cells irradiated with blue light under semi-aerobic conditions was higher than that with red light. Phosphatase treatment of the cell-free extracts strongly increased the DNA-binding affinity of the protein.

Base Sequence↗

PubMed1995

Sequencing and the alignment of structural genes in the nqr operon encoding the Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.

We previously cloned a part of nqr operon encoding the Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus [Hayashi et al., FEBS Lett. 356 (1994) 330-332]. From its nucleotide sequences, four consecutive open reading frames (ORF) encoding the gamma-subunit (27.7 kDa), two unidentified ORFs of 22.6 kDa and 21.5 kDa, and the beta-subunit (45.3 kDa) were recognized. The gene encoding the alpha-subunit was located upstream, and together with the recent report by Beattie et al. [FEBS Lett. 356 (1994) 333-338], the nqr operon was found to be constructed from six consecutive structural genes, where nqr1, nqr3 and nqr6 correspond to the alpha-, gamma-, and beta-subunits, respectively, of the NQR complex.

Amino Acid Sequence↗

PubMed1984

Rhodopseudomonas blastica atp operon. Nucleotide sequence and transcription.

The nucleotide sequence has been determined of a 12,368 base-pair region of DNA cloned from the non-sulphur photosynthetic bacterium Rhodopseudomonas blastica. It contains a cluster of six genes of which five encode the subunits of F1-ATPase; the sixth codes for an unknown protein. The genes are arranged in the same order as in the Escherichia coli unc operon, except that the unknown gene is placed between those for gamma and beta subunits. Neither the genes for F0 subunits, nor a homologue of the E. coli uncI gene is associated with this locus. The six genes are transcribed from a single promoter and we have designated this region the R. blastica atp operon. The two distal genes, beta and epsilon, may also be transcribed from a second promoter. Initiation and termination points for transcription have been identified by primer extensions and S1 nuclease mapping experiments. Signals involved in initiation of translation (Shine and Dalgarno sequences) and termination of transcription in the photosynthetic bacterium resemble those in E. coli. However, no common features can be identified in these two bacteria between 5' regions adjacent to sites of initiation of transcription. The sequence also contains a gene that encodes a protein homologous to discoidin, a cell surface lectin of Dictyostelium discoideum thought to be involved in cell--cell aggregation. Seven other reading frames have not been identified.

Amino Acid Sequence↗

PubMed1985

Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator.

Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Base Sequence↗

PubMed1985

Analysis of the requirements for transcription pausing in the tryptophan operon.

RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide. Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon. We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro. A model is presented that accounts for many of the findings obtained in this and other pausing studies.

Base Sequence↗

PubMed1986

Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase.

The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.

Endonucleases↗

PubMed1988

Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli.

Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes. We have shown previously that transcription of the ilvGMEDA operon in E. coli is greatly reduced in IHF mutants. We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion. These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator. The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF. These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.

Bacterial Proteins↗

PubMed1988

Structure and expression of the ompB operon, the regulatory locus for the outer membrane porin regulon in Salmonella typhimurium LT-2.

The ompB operon of Salmonella typhimurium encodes a positive transcriptional regulator OmpR and an inner membrane protein EnvZ. Both proteins are needed for the proper expression of the outer membrane proteins OmpC and OmpF. We have determined the nucleotide sequence of the ompB locus and its adjacent regions. A comparison between the S. typhimurium and Escherichia coli sequences revealed that the ompB locus is highly conserved. The sequence data also showed that ompR and envZ form an operon, where the coding regions overlap by four base-pairs. Utilizing ompR-lacZ and envZ-lacZ gene fusions, the translational levels of expression of these two genes were measured, showing that ompR is considerably more efficiently expressed than envZ. Analysis of ompR frameshift mutations showed that translation of envZ is almost totally dependent on the translation of the upstream gene ompR. The mechanism of this translational coupling appears to be a reinitiation of the ribosome at the overlapping region of the two genes. The characteristics of the OmpR and EnvZ proteins were in agreement with the known functions and cellular locations of these proteins. OmpR was found to contain a putative DNA binding site, while EnvZ contained two hydrophobic stretches typical of transmembrane regions. Both OmpR and EnvZ show extensive homologies with many proteins from a number of different origins, all of which function in pairs and through which environmental signals modulate gene expression. Hence, the tightly coupled synthesis of these proteins seems to be essential in eliciting a proper response in the transmembrane regulation of gene expression.

Amino Acid Sequence↗

PubMed1989

Ribosomal RNA operon anti-termination. Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms.

All Escherichia coli rrn operons show a common motif in which anti-terminator box B-box A sequences occur twice, first in the leader and again in the 16 S-23 S spacer. In this study we have analyzed several aspects of rrn anti-termination by leader and spacer anti-terminator sequences. Using DNA synthesis and a plasmid test system, we incorporated random changes into the leader anti-terminator region and examined these mutations for their ability to read through a strong terminator. We also examined anti-termination by synthetic box A and by rrn spacer region sequences. Information derived from these experiments was used to search the rrn sequences of other micro-organisms for possible anti-termination features. Our principal conclusions were that: (1) box A was sufficient for terminator readthrough; (2) we could show no positive requirement for box B in our test system; (3) many of the negative anti-terminator mutations caused a promoter up-effect in the absence of a terminator; (4) the search of rrn operons from other micro-organisms revealed that anti-terminator-like box B-box A sequences exist in leader and spacer regions of both eubacteria and archaebacteria. The frequent occurrence of this pattern suggested that the E. coli rrn anti-termination motif is widespread in nature and has been conserved in microbial evolution.

Base Sequence↗

PubMed1989

Ribosomal affinity and translational initiation in Escherichia coli. In vitro investigations using translational initiation regions of differing efficiencies from the atp operon.

The atp operon of Escherichia coli comprises nine genes that are differentially expressed. The control of the atp genes' expression rates has been shown to be exercised primarily at the level of translational initiation, but how is this achieved in molecular terms? In order to study the interactions of 30 S ribosomal subunits with specifically the translational initiation regions (TIRs) of atpB, atpE and atpG, restriction fragments bearing these TIRs were excised from the atp operon and cloned into an SP6 promoter transcription vector. mRNA transcripts were made in vitro and used in primer extension inhibition studies and equilibrium mRNA-30 S ribosomal subunit binding measurements. The binding of 30 S ribosomal subunits blocked primer extension 14 to 15 bases downstream from the respective translational start codons. The affinities of binding of 30 S ribosomal subunits showed the relationship atpE greater than atpB greater than atpG. This was also the order of the efficiency of translation promoted by the respective TIRs, both in vivo and on the in vitro synthesized mRNA fragments. Thus, the affinity of 30 S ribosomal subunits is at least to some extent correlated with the rate of translational initiation.

Base Sequence↗

PubMed1989

Organization and structure of the Methanococcus transcriptional unit homologous to the Escherichia coli "spectinomycin operon". Implications for the evolutionary relationship of 70 S and 80 S ribosomes.

By means of an immunological approach and a subsequent chromosome-walking strategy a chromosomal region encoding ribosomal proteins in the archaebacterium Methanococcus vannielii was cloned. The determination of the nucleotide sequence of the 7.8 x 10(3) base DNA fragment revealed the existence of 14 putative ribosomal protein genes and two unidentified open reading frames. They are organized in a transcriptional unit that is very similar to the Escherichia coli "spectinomycin operon" in respect of both gene composition and gene order. The Methanococcus transcriptional unit contains, in addition to those genes whose products have a homologue in the E. coli operon, three genes whose products share sequence similarity with eukaryotic 80 S but not with eubacterial ribosomal proteins. The Methanococcus ribosomal proteins almost exclusively exhibit a higher sequence similarity to eukaryotic 80 S ribosomal proteins than to those of eubacteria and many of them have a size intermediate between those of their eukaryotic and eubacterial homologues. These results are discussed in terms of a hypothesis that implies that the recent eubacterial ribosome developed by a "minimization" process from a more complex organelle and that the archaebacterial ribosome has maintained features of this ancestor.

Archaea↗

PubMed1989

Genetic structure of the bacteriophage P22 PL operon.

The sequence of 1416 base-pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combination from Plac UV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to lambda ral and cIII, respectively. P22 kil, like lambda kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and lambda kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream from erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the lambda red genes from plasmids. Sequences that include the stop codon for gene 17 may form a small stem-loop structure and are nearly identical to lambda sequences that contain the stop codon for ssb, which is near lambda tL 2b. Plasmids that include the P22 structure negatively regulate kil gene expression in cis.

Amino Acid Sequence↗

PubMed1990

Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon.

This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

Amino Acid Sequence↗

PubMed1991

Specificity of antitermination mechanisms. Suppression of the terminator cluster T1-T2 of Escherichia coli ribosomal RNA operon, rrnB, by phage lambda antiterminators.

Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region. This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at the end of an rrn operon, such as rrnB. We have tested the specificity with which the T1-T2 terminators override an antitermination mechanism, by placing the terminator cluster downstream from the nut and qut sites recognized by phage lambda N and Q gene antiterminators, respectively. Measurement of downstream gene expression shows that RNA polymerase modified by either N or Q reads through the T1-T2 terminators quite efficiently. This supports the view that T1-T2 are not superterminators, and that the rrn antitermination mechanism may have a restricted terminator specificity.

Bacteriophage lambda↗

PubMed1992

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20.

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

Bacterial Proteins↗

PubMed1992

Synthesis and degradation of the mRNA of the Tn21 mer operon.

The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (merTn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg2+ and merD, which may also play a minor regulatory role. Comparison of the relative abundance of the 5' and 3' ends of the merTPCAD transcript revealed a strong transcriptional gradient in the operon, consistent with previous observations of lower relative abundance of the more promoter-distal gene products. In vivo mRNA degradation rates varied only slightly for the different genes: however, the rates of mRNA synthesis varied considerably from the beginning to the end of the operon. Specifically, mRNA corresponding to the promoter-proximal genes, merTPC, achieved a maximum in vivo synthesis rate between 60 and 120 seconds after induction; this rate was maintained for approximately ten minutes. In contrast, the synthesis rates of mRNA corresponding to the promoter-distal genes merA and merD, were initially fivefold lower than the rates of the promoter-proximal genes for the first five minutes after induction, and then rose gradually to approximately 50% of the merTPC synthesis rates. These data suggested that early after induction only 20% of the transcripts initiating at merT proceed beyond merC. At later times after induction approximately 50% of the transcripts proceed beyond merC. Nuclease end mapping did not reveal any discrete termination events in the merPCA region, thus, premature termination may occur at many sites.

Base Sequence↗

PubMed1992

RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa.

Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.

Amino Acid Transport Systems↗