Σ70 Promoter Research Articles

Chloroplast transcription, untangling the Gordian Knot.

Chloroplast transcription, untangling the Gordian Knot.

Assessing the effects of data selection and representation on the development of reliable E. coli sigma 70 promoter region predictors.

As the number of sequenced bacterial genomes increases, the need for rapid and reliable tools for the annotation of functional elements (e.g., transcriptional regulatory elements) becomes more desirable. Promoters are the key regulatory elements, which recruit the transcriptional machinery through binding to a variety of regulatory proteins (known as sigma factors). The identification of the promoter regions is very challenging because these regions do not adhere to specific sequence patterns or motifs and are difficult to determine experimentally. Machine learning represents a promising and cost-effective approach for computational identification of prokaryotic promoter regions. However, the quality of the predictors depends on several factors including: i) training data; ii) data representation; iii) classification algorithms; iv) evaluation procedures. In this work, we create several variants of E. coli promoter data sets and utilize them to experimentally examine the effect of these factors on the predictive performance of E. coli σ 70 promoter models. Our results suggest that under some combinations of the first three criteria, a prediction model might perform very well on cross-validation experiments while its performance on independent test data is drastically very poor. This emphasizes the importance of evaluating promoter region predictors using independent test data, which corrects for the over-optimistic performance that might be estimated using the cross-validation procedure. Our analysis of the tested models shows that good prediction models often perform well despite how the non-promoter data was obtained. On the other hand, poor prediction models seems to be more sensitive to the choice of non-promoter sequences. Interestingly, the best performing sequence-based classifiers outperform the best performing structure-based classifiers on both cross-validation and independent test performance evaluation experiments. Finally, we propose a meta-predictor method combining two top performing sequence-based and structure-based classifiers and compare its performance with some of the state-of-the-art E. coli σ 70 promoter prediction methods.

Promoter Prediction in Bacterial DNA Sequences Using Expectation Maximization and Support Vector Machine Learning Approach

Promoter is a part of the DNA sequence that comes before the gene and is key as a regulator of genes. Promoter prediction helps determine gene position and analyze gene expression. Hence, it is of great importance in the field of bioinformatics. In bioinformatics research, a number of machine learning approaches are applied to discover new meaningful knowledge from biological databases. In this study, two learning approaches, expectation maximization clustering and support vector machine classifier (EMSVM) are used to perform promoter detection. Expectation maximization (EM) algorithm is used to identify groups of samples that behave similarly and dissimilarly, such as the activity of promoters and non-promoters in the first stage, while the support vector machine (SVM) is used in the second stage to classify all the data into the correct class category. We have applied this method to datasets corresponding to σ24, σ32, σ38, σ70 promoters and its effectiveness was demonstrated on a range of different promoter regions. Furthermore, it was compared with other classification algorithms to indicate the appropriate performance of the proposed algorithm. Test results show that EMSVM performs better than other methods.

Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks.

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions (< 1.3-fold). These gates were connected to build larger circuits, including the Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).

Development of a tool for computational prediction of σ70 promoters in Pseudomonas spp using SVM and HMM approaches

Promoters are regions in DNA that play important role in the regulation of gene expression. The ability to locate promoters within a section of DNA is known to be a very difficult and important task in DNA analysis. Since experimental techniques to identify promoters are costly and time consuming, in silico methods offer an alternative. In this study, we have developed a tool for identification of s70 promoters in the –10 and –35 regions of sequences from Pseudomonas spp. Promoters were predicted using both Support Vector Machine (SVM) and Hidden Markov Model (HMM) based approaches. SVM performed better when trained using RBF kernel with a cross-validation of 5 and a value of 0.03 for the gamma parameter. The module developed using SVM showed a sensitivity of 78% and a specificity of 80%. The programmes required to process the user input were written using Perl and HTML codes were used to create a user interface. The user interface accepts a query sequence and the processed result will be displayed in a new window. The tool named ‘PROMIT’ (PROMoter Identification Tool), was developed in the Windows platform, has a user friendly interface and works well for sequences from Pseudomonas spp.

Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

BackgroundHeterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products.MethodologyWe have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways.ConclusionsWe have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products.

Genomic analysis of the regulatory elements and links with intrinsic DNA structural properties in the shrunken genome of Buchnera

BackgroundBuchnera aphidicola is an obligate symbiotic bacterium, associated with most of the aphididae, whose genome has drastically shrunk during intracellular evolution. Gene regulation in Buchnera has been a matter of controversy in recent years as the combination of genomic information with the experimental results has been contradictory, refuting or arguing in favour of a functional and responsive transcription regulation in Buchnera.The goal of this study was to describe the gene transcription regulation capabilities of Buchnera based on the inventory of cis- and trans-regulators encoded in the genomes of five strains from different aphids (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, Cinara cedri and Cinara tujafilina), as well as on the characterisation of some intrinsic structural properties of the DNA molecule in these bacteria.ResultsInteraction graph analysis shows that gene neighbourhoods are conserved between E. coli and Buchnera in structures called transcriptons, interactons and metabolons, indicating that selective pressures have acted on the evolution of transcriptional, protein-protein interaction and metabolic networks in Buchnera. The transcriptional regulatory network in Buchnera is composed of a few general DNA-topological regulators (Nucleoid Associated Proteins and topoisomerases), with the quasi-absence of any specific ones (except for multifunctional enzymes with a known gene expression regulatory role in Escherichia coli, such as AlaS, PepA and BolA, and the uncharacterized hypothetical regulators YchA and YrbA). The relative positioning of regulatory genes along the chromosome of Buchnera seems to have conserved its ancestral state, despite the genome erosion. Sigma-70 promoters with canonical thermodynamic sequence profiles were detected upstream of about 94% of the CDS of Buchnera in the different aphids. Based on Stress-Induced Duplex Destabilization (SIDD) measurements, unstable σ70 promoters were found specifically associated with the regulator and transporter genes.ConclusionsThis genomic analysis provides supporting evidence of a selection of functional regulatory structures and it has enabled us to propose hypotheses concerning possible links between these regulatory elements and the DNA-topology (i.e., supercoiling, curvature, flexibility and base-pair stability) in the regulation of gene expression in the shrunken genome of Buchnera.

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

In Legionella pneumophila, the regulation of the flagellum and the expression of virulence traits are linked. FleQ, RpoN and FliA are the major regulators of the flagellar regulon. We demonstrated here that all three regulatory proteins mentioned (FleQ, RpoN and FliA) are necessary for full in vivo fitness of L. pneumophila strains Corby and Paris. In this study, we clarified the role of FleQ for fliA expression from the level of mRNA toward protein translation. FleQ enhanced fliA expression, but FleQ and RpoN were not necessary for basal expression. In addition, we identified the initiation site of fliA in L. pneumophila and found a putative σ(70) promoter element localized upstream. The initiation site was not influenced in the ΔfleQ or ΔrpoN mutant strain. We demonstrated that there is no significant difference in the regulation of fliA between strains Corby and Paris, but the FleQ-dependent induction of fliA transcription in the exponential phase is stronger in strain Paris than in strain Corby. In addition, we showed for the first time the presence of a straight hook at the pole of the non-flagellated ΔfliA and ΔfliD mutant strains by electron microscopy, indicating the presence of an intact basal body in these strains.

Genome-wide detection of novel regulatory RNAs in E. coli

The intergenic regions in bacterial genomes can contain regulatory leader sequences and small RNAs (sRNAs), which both serve to modulate gene expression. Computational analyses have predicted the presence of hundreds of these noncoding regulatory RNAs in Escherichia coli; however, only about 80 have been experimentally validated. By applying a deep-sequencing approach, we detected and quantified the vast majority of the previously validated regulatory elements and identified 10 new sRNAs and nine new regulatory leader sequences in the intergenic regions of E. coli. Half of the newly discovered sRNAs displayed enhanced stability in the presence of the RNA-binding protein Hfq, which is vital to the function of many of the known E. coli sRNAs. Whereas previous methods have often relied on phylogenetic conservation to identify regulatory leader sequences, only five of the newly discovered E. coli leader sequences were present in the genomes of other enteric species. For those newly identified regulatory elements having orthologs in Salmonella, evolutionary analyses showed that these regions encoded new noncoding elements rather than small, unannotated protein-coding transcripts. In addition to discovering new noncoding regulatory elements, we validated 53 sRNAs that were previously predicted but never detected and showed that the presence, within intergenic regions, of σ(70) promoters and sequences with compensatory mutations that maintain stable RNA secondary structures across related species is a good predictor of novel sRNAs.

A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti

BackgroundSmall untranslated RNAs (sRNAs) are widespread regulators of gene expression in bacteria. This study reports on a comprehensive screen for sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti applying deep sequencing of cDNAs and microarray hybridizations.ResultsA total of 1,125 sRNA candidates that were classified as trans-encoded sRNAs (173), cis-encoded antisense sRNAs (117), mRNA leader transcripts (379), and sense sRNAs overlapping coding regions (456) were identified in a size range of 50 to 348 nucleotides. Among these were transcripts corresponding to 82 previously reported sRNA candidates. Enrichment for RNAs with primary 5'-ends prior to sequencing of cDNAs suggested transcriptional start sites corresponding to 466 predicted sRNA regions. The consensus σ70 promoter motif CTTGAC-N17-CTATAT was found upstream of 101 sRNA candidates. Expression patterns derived from microarray hybridizations provided further information on conditions of expression of a number of sRNA candidates. Furthermore, GenBank, EMBL, DDBJ, PDB, and Rfam databases were searched for homologs of the sRNA candidates identified in this study. Searching Rfam family models with over 1,000 sRNA candidates, re-discovered only those sequences from S. meliloti already known and stored in Rfam, whereas BLAST searches suggested a number of homologs in related alpha-proteobacteria.ConclusionsThe screening data suggests that in S. meliloti about 3% of the genes encode trans-encoded sRNAs and about 2% antisense transcripts. Thus, this first comprehensive screen for sRNAs applying deep sequencing in an alpha-proteobacterium shows that sRNAs also occur in high number in this group of bacteria.

An Abortive Isomerization Branch in the Transcription Initiation Pathway At a σ54 Promoter As Revealed By Single Molecule Fluorescence Microscopy

Regulated transcription initiation is a complex process that involves multiple protein factors and a series of polymerase-DNA complexes that are intermediates in the reaction. This complexity presents a significant challenge for ensemble experiments that aim to elucidate the reaction pathway. We here report the kinetic mechanism of initiation at the σ54 promoter of the glnALG operon in Salmonella typhimurium. This prototypical activator-dependent promoter is regulated by nitrogen stress. To circumvent the complexity of ensemble analysis, we used multi-wavelength single-molecule fluorescence colocalization methods to follow initiation reactions on individual surface-anchored DNA molecules that contain σ54 promoters. Three distinguishable dye labels enabled us to follow reactions in which RNA polymerase binding, open complex formation, escape into transcription elongation and departure of the σ54 subunit were detected in individual transcription complexes, and the interconversion kinetics for all states were measured. Transcription initiation from this promoter occurs only following a polymerase isomerization that is induced by interaction with the NtrC activator protein in the presence of ATP. However, we observed that with NtrC present the polymerase leaves the promoter faster than the combined rates of initiation plus closed complex departure. Thus, a fraction of activator-mediated polymerase isomerizations displace the polymerase from the promoter without initiating transcript synthesis. This activator-induced abortive isomerization is a non-productive branch off of the initiation pathway and is more common than productive transcription initiation. We speculate that abortive isomerization is a consequence of the large energy input required to disrupt promoter-polymerase interactions prior to promoter escape. Taken together, our results define the full pathway and dynamics of initiation at this activator-dependent promoter and illustrate the power of multi-wavelength single-molecule colocalization methods in the elucidation of complex biological regulatory mechanisms.

Identification of the transcription factor responsible for l-malate-dependent regulation in the marine Antarctic bacteriumPseudoalteromonas haloplanktisTAC125

Two-component systems are widespread in nature and constitute the most common mechanism of transmembrane signal transduction in bacteria. Recently, a functionally active two-component system consisting of malS and malR genes possibly involved in the expression of a C4-dicarboxylate transporter system (dctAB operon) was identified in the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. In this paper, we performed a functional analysis of the two-component system and demonstrated its involvement in the regulation of the expression of C4-dicarboxylate transporter genes. The expression of the C4-dicarboxylate transporter genes was induced by l-malate with the promoter element located upstream of the dctA gene being active only in the presence of the inducer. A sigma(54) promoter responsible for the l-malate dependent transcription regulation was identified and functionally characterized. The molecular mechanism involves an inverted repeat sequence located upstream the sigma(54) promoter that was shown to bind regulatory proteins only in the presence of l-malate. The protein factor responsible for the induction of the dctAB operon expression was eventually identified as the transcriptional regulatory protein MalR. MalR is the first transcriptional factor identified in P. haloplanktis TAC125 and one of the few transcriptional modulators reported so far in cold adapted bacteria.

Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath

A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XylE) or beta-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) sigma(54) promoter or particulate methane monooxygenase (pMMO) sigma(70) promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When beta-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis.

Campylobacter lari: molecular and comparative analyses of the virulence-associated chromosome locus J (vacJ) gene homologue, including the promoter region

Following TA cloning and sequencing with a novel in silicodesigned polymerase chain reaction (PCR) primer pair (f-ClvacJ/r-ClvacJ), approximately 750 base pairs (bp) of promoter and structural gene regions for vacJ and its adjacent genetic loci (approximately 1.14 kbp) were identified in 20 isolates of Campylobacter lari (urease-negative C. lari [n=7]; urease-positive thermophilic Campylobacter [n=13]). The nucleotide sequences of an approximately 70-bp non-coding region, including the typical promoter structure, showed sequence differences at 12 loci among 21 isolates including C. lari RM2100. The putative σ70 promoter region upstream of the putative open reading frame (ORF), a start codon TTG and a probable ribosome binding site, AGGA, for the vacJ gene were also identified in all 21 C. lari isolates examined. Each ORF for the vacJ terminated with a TAA stop codon. No hypothetical transcriptional terminators were identified within the amplicons. The putative ORFs of the vacJ gene from 21 C. lari isolates consisted of 684 bases, similarly differing from those of the other thermophilic campylobacters (696 bases for C. jejuni RM1221 and NCTC11168 and C. coli RM2228; 690 for C. upsaliensis RM3195). Reverse transcription PCR analysis confirmed the transcription of the vacJ gene in the C. lari cells. A neighbour joining tree suggested a strong molecular discrimination efficacy between UPTC and UN C. lari employing vacJ nucleotide sequence information. The vacJ gene homologue from C. lari organisms appears not to be a lipoprotein signal peptide or a signal peptide in silico.

Bioinformatics Resources for the Study of Gene Regulation in Bacteria

Bioinformatics Resources for the Study of Gene Regulation in Bacteria

Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase

AAA+ proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA+ characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA+ domain, PspF1–275, remodels the bacterial σ54–RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF1–275 indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent α-helical α/β subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA+ proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA+ ring. Homotropic coordination is functionally important to remodel the σ54 promoter. We propose a structural symmetry-based model for homotropic control in the AAA+ characteristic ring architecture.

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).

Mapping ATP-dependent Activation at a σ54 Promoter

The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-sigma(54)-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with down-stream melted DNA.

The recognition and prediction of σ70 promoters in Escherichia coli K-12

Based on the conservation analysis of the 683 latest experimentally verified σ 70-promoter sequences of Escherichia coli K-12, it is found that the conservative hexamers segments in different sites play a key role of promoter regions, a novel position-correlation scoring matrix (PCSM) algorithm for predicting σ 70 promoter is presented. The predictive capacity of the algorithm is tested by 10-cross validation test. The results show that the overall prediction accuracies (sensitivity) and specificity are 91% and 81%, respectively. By selecting the 683 experimentally verified σ 70 promoters as training set and searching for the complete sequence in E. coli K-12 with 4639221 bp. Results show that the 100% of the 683 experimentally verified σ 70 promoters have been identified and some possible promoters are predicted.

The Upstream-activating Sequences of the σ54 Promoter Pu of Pseudomonas putida Filter Transcription Readthrough from Upstream Genes

Although the m-xylene-responsive sigma54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive sigma54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of lambda phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon.