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PubMed2003

The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase.

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E. coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm. The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo. Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T. pallidum encounters high Zn2+ levels.

ATP-Binding Cassette Transporters↗

PubMed1996

The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei.

The D-alanine incorporation system allows Lactobacillus casei to modulate the chemical properties of lipoteichoic acid (LTA) and hence control its proposed functions, i.e., regulation of autolysin action, metal ion binding, and the electromechanical properties of the cell wall. The system requires the D-alanine-D-alanyl carrier protein ligase (Dcl) and the D-alanyl carrier protein (Dcp). Our results indicate that the genes for these proteins are encoded in the dlt operon and that this operon contains at least 2 other genes, dltB and dltD. The aim of this paper is to describe the genetic organization of the operon, the role of the D-alanyl carrier protein, and the function of the putative protein encoded by dltB in the intramembranal translocation of the activated D-alanine.

Carrier Proteins↗

PubMed2005

Characterization of a cmeABC operon in a quinolone-resistant Campylobacter coli isolate of Irish origin.

In this study, a tripartite-operon-encoding efflux system together with its regulatory gene was characterized in an Irish Campylobacter coli isolate CIT-382 showing high-level resistance to nalidixic acid and ciprofloxacin. Sequence comparisons revealed significant homology between C. coli and the cmeABC operon of Campylobacter jejuni. Conservation of functional sequence domains and motifs were noted among C. coli and similar operons in unrelated organisms. A transcriptional regulatory gene cmeR located proximal to cmeABC was also identified. C. coli CIT-382 harbored the Thr-86-Ile amino acid substitution in the gyrA gene. Accumulation studies with ethidium bromide in the presence of known efflux pump inhibitors confirmed the presence of efflux pump activity in C. coli CIT-382. The efflux pump inhibitor PAbetaN had no effect on the MICs to quinolones. Our data suggest that the gyrA gene mutation is the main contributor to the high-level nalidixic acid and ciprofloxacin resistance observed in this Irish C. coli CIT-382 isolate.

Anti-Bacterial Agents↗

PubMed2004

Operons in eukaryotes.

It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

Alternative Splicing↗

PubMed2002

Identifying operons and untranslated regions of transcripts using Escherichia coli RNA expression analysis.

Microarrays traditionally have been used to assay the transcript expression of coding regions of genes. Here, we use Escherichia coli oligonucleotide microarrays to assay transcript expression of both open reading frames (ORFs) and intergenic regions. We then use hidden Markov models to analyse this expression data and estimate transcription boundaries of genes. This approach allows us to identify 5' untranslated regions (5' UTRs) of transcripts as well as genes that are likely to be operon members. The operon elements we identify correspond to documented operons with 99% specificity and 63% sensitivity. Similarly we find that our 5' UTR results accurately coincide with experimentally verified promoter regions for most genes.

5' Untranslated Regions↗

PubMed1998

Structural characterization of the virB operon on the hairy-root-inducing plasmid A4.

The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferring tumorigenic symptoms on plants upon infection by its host bacterium, Agrobacterium rhizogenes. The virB operon on pRiA4 has been sequenced and found to be composed of 11 genes, virB1 to virB11, whose products mostly appear to be associated with the cell membrane. A novel structural characteristic is frequent overlappings between the translation termination and initiation codons of adjacent genes. This is indicative of fine tuning of relative translation frequencies for each VirB protein. These results support the view that VirB multisubunit complexes provide facilities for T-DNA transfer at the bacterial cell membrane. The structural organization of the pRiA4 virB operon was essentially identical to that of the previously reported virB operons of tumor-inducing plasmids, pTiC58 and pTiA6, and the corresponding VirB proteins of the three plasmids were extremely homologous to one another. On the basis of the structural similarity of each VirB protein, the phylogenetic relationship among pRiA4, pTiC58, and pTiA6 is discussed.

Bacterial Proteins↗

PubMed1998

Mechanism of translational coupling in the nifLA operon of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae encodes the sensor-activator pair involved in the regulation of other nif genes. Balanced synthesis of both proteins, which is required for correct regulation, is achieved by coupling translation of nifA to that of nifL. The mechanism of translational coupling at the nifLA operon was analysed using a specialized ribosome system, and the effect of substituting the natural Shine-Dalgarno of nifL or nifA for specialized Shine-Dalgarno sequences was determined. Our results indicate that translational coupling occurs in this operon by a reinitiation mechanism. Additionally, reinitiation at the nifA can happen even in the absence of good Shine-Dalgarno recognition by the reinitiating ribosome, although its efficiency is lower. The effect of a putative translational enhancer sequence (downstream box) on translational coupling efficiency was also determined. Mutations that reduce the homology of the putative downstream box to the consensus had only a minor effect on nifA translation by wild-type ribosomes. However, they had a significant effect on nifA translation by specialized ribosomes, suggesting that recognition of the downstream box may compensate inefficient ribosomal interactions with the Shine-Dalgarno sequence.

Bacterial Proteins↗

PubMed2002

Transcriptional regulation of fis operon involves a module of multiple coupled promoters.

The transcription of the Escherichia coli fis gene is strongly activated during the outgrowth of cells from stationary phase. The high activity of the promoter of the fis operon requires the transcription factor IHF. Previously, we identified a divergent promoter, div, located upstream of the fis promoter. In this study we demonstrate that at least two additional promoters, designated fis P2 and fis P3, are located in the control region of the fis operon. The fis P2 and div promoters overlap completely, whereas fis P3 and div P are arranged as face-to-face divergent promoters. We show that the div and the tandem fis promoters counterbalance each other, such that their activity is kept on a lower than potentially attainable level. Furthermore, we demonstrate an unusual activation mechanism by IHF, involving a coordinated shift in the balance of promoter activities. We infer that these coupled promoters represent a regulatory module and propose a novel "dynamic balance" mechanism involved in the transcriptional control of the fis operon.

Base Sequence↗

PubMed1996

Selfish operons: horizontal transfer may drive the evolution of gene clusters.

A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa. Bacterial operons are typically composed of genes whose products contribute to a single function. If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift. From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer. The probability of transfer of multiple genes increases when genes are physically proximate. From a gene's perspective horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions. Since organism bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes. The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host. This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions.

Alleles↗

PubMed1983

DNA sequence of the tandem ribosomal RNA promoter for B. subtilis operon rrnB.

A new ribosomal RNA operon designated rrnB has been identified by screening a Charon 4a library of cloned B. subtilis sequences. Clones containing the promoter region of this operon are unstable in E. coli unless a special vector possessing a transcriptional terminator is used. DNA sequence data suggests that this operon contains two tandem putative promotor regions not unlike those found in E. coli. There are 92 base pairs separating the two "-10 regions" of the promotors. The second is 180 bp upstream from the start site for mature 16S RNA. A potential 29 base pair stem structure necessary for processing of the mature 16S RNA sequence can also be predicted from this analysis.

Bacillus subtilis↗

PubMed1984

Interaction of cyclic AMP receptor protein with the ilvB biosynthetic operon in E. coli.

DNase and restriction site protection studies show that cAMP and its receptor protein (CRP) bind to the promoter of the ilvB operon at approximately position -44 to -82. This region contains sequences that are homologous to those found in other CRP-dependent promoters. In vitro transcription from the ilvB promoter was markedly increased by the addition of cAMP and CRP. This stimulation was not found when the ilvB template lacked the proposed CRP binding site. cAMP-CRP did not alter the extent of transcription termination within the ilvB leader suggesting that this regulatory system may be independent of the attenuation mechanism involved in the negative control of this operon. The results of restriction enzyme site protection studies and experiments with altered promoter fragments indicate that the mechanism for CRP stimulation of the ilvB operon may be similar to a model recently proposed for lac.

Acetolactate Synthase↗

PubMed1985

Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli.

In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome. One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase. We report here the complete 3.0 kb nucleotide sequence of the alpha operon. In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon.

Amino Acid Sequence↗

PubMed1985

Structure of the Escherichia coli S10 ribosomal protein operon.

The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.

Amino Acid Sequence↗

PubMed1987

Gene organization, transcription signals and processing of the single ribosomal RNA operon of the archaebacterium Thermoproteus tenax.

The single ribosomal RNA (rRNA) operon from the extreme thermophile and archaebacterium Thermoproteus tenax was sequenced. Sites of transcriptional initiation and termination were established and the processing sites on the primary transcript were mapped with nuclease S1. The operon contained genes coding for 16S and 23S RNAs but lacked those coding for tRNA and 5S RNA. Transcription initiates 175 bp upstream from the start of the 16S RNA gene (Wich et al., EMBO J. 6, 523-528, 1987) and terminates 49 bp downstream from the 23S RNA gene within a long pyrimidine sequence. An open reading frame downstream from the rRNA operon is transcribed. The sequences bordering both 16S and 23S RNA genes can form putative processing stems in the primary transcript that involve the whole of the 16S-23S RNA spacer. The stems contain irregular features that constitute processing signals and are conserved in other archaebacteria. The 16S RNA stem is cut prior to that of the 23S RNA and RNA maturation follows. An unusual 14 bp helix can form between the extremities of the transcript such that the whole transcript is highly structured and a fork-like structure is formed together with the processing stems. The 23S RNA sequence was aligned with other available 23S-like RNA sequences (Leffers et al., J. Mol. Biol. 195, in press): a putative secondary structure exhibiting archaebacterial-specific features was deduced using comparative sequence analyses. A rooted phylogenetic tree was also derived for the archaebacteria that confirms their division into three major subgroups.

Archaea↗

PubMed1987

Molecular characterization of the virD operon from Agrobacterium tumefaciens.

The Agrobacterium tumefaciens Ti plasmid virulence (vir) region contains at least six transcriptional units required for the efficient transfer of T-DNA to the plant genome (virA, B, C, D, E, and G). We have reported that two proteins encoded by the 5'portion of the virD operon are required for a site-specific endonuclease activity that nicks the direct repeats which flank the T-DNA. We have presented the nucleotide sequence for this portion of the operon. The nucleotide sequence of the remainder of the virD operon essential for virulence has now been determined. Two additional open reading frames encode proteins of 21.3 and 75.8 kilodaltons (kd). Translational fusions between virD2, virD3, and virD4 proteins and trpE produced fusion proteins of the size predicted from the nucleotide sequence data. We have used antisera directed against the trpE-virD2 fusion protein to detect both native virD2 protein and a virD2-lacZ fusion protein in crude extracts from Agrobacterium.

Amino Acid Sequence↗

PubMed1987

Transcription of the tRNA-tufB operon of Escherichia coli: activation, termination and antitermination.

Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping. TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions. Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB transcription. This demonstrates the presence of a region, upstream of the tRNA-tufB promoter, that enhances the expression of the operon. DNA fragments bearing this upstream activator region do not display an abnormal electrophoretic mobility, as has been observed for the rrnB P1 upstream activator. Deletions starting in the first tRNA gene and directing towards tufB reveal at least two sites that influence tufB transcription. One signals transcription termination in the intergenic region between thrT and tufB. The other may be involved in antitermination. Possible mechanisms underlying antitermination and termination are considered in the light of the nucleotide sequence.

Base Sequence↗

PubMed1987

The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12.

In this report we present the complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli. This operon contains five genes encoding four of the five enzymes required for the biosynthesis of isoleucine and valine. We identify and describe the coding regions for these five structural genes and the structural and functional features of the flanking and internal regulatory regions of this operon. This new information contributes to a more complete understanding of the overall control of the biosynthesis of isoleucine and valine.

Amino Acid Sequence↗

PubMed1988

Regulation of in vitro expression of the Escherichia coli frd operon: alanine and Fnr represent positive and negative control elements.

The frdABCD operon of Escherichia coli encodes the anaerobically expressed terminal electron transport enzyme, fumarate reductase. Two mutually exclusive hairpin loop structures can occur in frdmRNA just downstream of the start of the frdA cistron. The mRNA sequence involved encodes a stretch of sequence rich in Ala and uses all four of the codons for this amino acid. In vitro expression of the frdABCD operon showed that as the level of plasmid DNA was increased from 150 fmol to 225 fmol, transcription of mRNA was suddenly elevated 6.5-fold, consistent with the concept of titrating out a repressor protein. Further studies showed that the concomitant 10.9-fold increase in translation of protein was heavily biased towards the proximal end of the operon, with little or no expression of FrdC or FrdD and a ratio of FrdA:FrdB of 2.6:1. Addition of Ala to the S-30 extract caused a 6.1-fold amplification of frd messenger transcription, a 17.6-fold increase in Frd protein translation, and a balancing of the subunit ratios to 1:1:1:1. The expression of the bla gene carried on the plasmid was not affected by DNA titration or the addition of Ala. When fnr DNA was added in equimolar ratio to frdDNA the amplification of fumarate reductase expression by Ala was abolished and the ratio of subunits produced showed a high degree of polarity with or without Ala.

Alanine↗