An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli

Fernando Macián, Ignacio Pérez-Roger, M. Eugenia Armengod

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39 Scopus citations

Abstract

We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the β-galactosidase (βGal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy λ prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

Original languageEnglish (US)
Pages (from-to)17-24
Number of pages8
JournalGene
Volume145
Issue number1
DOIs
StatePublished - Jul 22 1994
Externally publishedYes

Keywords

  • Recombinant DNA
  • gene fusions
  • promoter detection
  • transcriptional terminators

ASJC Scopus subject areas

  • Genetics

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