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Visualizing the activity of Escherichia coli divergent promoters and probing their dependence on superhelical density using dual-colour fluorescent reporter vector.

Masulis IS, Babaeva ZSh, Chernyshov SV, Ozoline ON - Sci Rep (2015)

Bottom Line: Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes.RNA product transcribed in antisense direction is suggested as a novel RNA.Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of functional genomics and cellular stress, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290, Russian Federation.

ABSTRACT
Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

No MeSH data available.


Related in: MedlinePlus

Scheme illustrating successive steps in the construction of pPF1.
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f1: Scheme illustrating successive steps in the construction of pPF1.

Mentions: Bidirectional promoter probe vectors have been proposed as a useful tool for divergent transcription studying about twenty years ago. The first systems exploited genes of different enzymes with easily detectable activity, such as β-galactosidase, alkaline phosphatase or bacterial luciferase1920. The widespread use of fluorescent proteins has created the basis for a new generation of reporter systems. They have advantage of simultaneous measurements of both reporter genes expression using identical rather then enzyme-specific modes of registration. Here we introduce the dual-colour vector derived from the pET-28b plasmid and supplemented with two genes encoding fluorescent proteins EGFP and mCherry. Integrated genes were PCR-amplified from plasmids pEGFP-N3 and pmCherry-C1 (Fig. 1) using one and the same pair of primers (GFP-NdeI and GFP-Xho) since 22 bp in their C-terminal parts, and 21 bp of N-termini are identical. As a result, two intermediate plasmids - pET28-EGFP and pET28-mCherry were obtained (Fig. 1).


Visualizing the activity of Escherichia coli divergent promoters and probing their dependence on superhelical density using dual-colour fluorescent reporter vector.

Masulis IS, Babaeva ZSh, Chernyshov SV, Ozoline ON - Sci Rep (2015)

Scheme illustrating successive steps in the construction of pPF1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4469952&req=5

f1: Scheme illustrating successive steps in the construction of pPF1.
Mentions: Bidirectional promoter probe vectors have been proposed as a useful tool for divergent transcription studying about twenty years ago. The first systems exploited genes of different enzymes with easily detectable activity, such as β-galactosidase, alkaline phosphatase or bacterial luciferase1920. The widespread use of fluorescent proteins has created the basis for a new generation of reporter systems. They have advantage of simultaneous measurements of both reporter genes expression using identical rather then enzyme-specific modes of registration. Here we introduce the dual-colour vector derived from the pET-28b plasmid and supplemented with two genes encoding fluorescent proteins EGFP and mCherry. Integrated genes were PCR-amplified from plasmids pEGFP-N3 and pmCherry-C1 (Fig. 1) using one and the same pair of primers (GFP-NdeI and GFP-Xho) since 22 bp in their C-terminal parts, and 21 bp of N-termini are identical. As a result, two intermediate plasmids - pET28-EGFP and pET28-mCherry were obtained (Fig. 1).

Bottom Line: Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes.RNA product transcribed in antisense direction is suggested as a novel RNA.Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of functional genomics and cellular stress, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290, Russian Federation.

ABSTRACT
Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

No MeSH data available.


Related in: MedlinePlus