An antisense RNA-mediated transcriptional attenuation mechanism functions in Escherichia coli

Abstract

Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

FIG. 1.

Schematic linear maps of the plasmids used in this study. The linear plasmids and genes and sites contained within them are not drawn to scale. Genes and sites of importance are highlighted as follows: replicon type, blue; selectable marker genes, yellow; rep genes or gene fragments, red; antisense RNA gene, light red; lacZ reporter gene, purple. Zigzag lines, in-frame (translational) fusions between rep and lacZ. Premature stop codons (TAA) separate the rep and lac reading frames, with the latter containing translational start signals. Some promoters are indicated by arrows. Inactivated promoters are crossed over. Approximate positions of attenuators (Att) and rrnB terminators (rrnB-T) are shown. For more details, see the text.

FIG. 2.

In vivo attenuation patterns in E. coli. An analysis of RNAs encoded by the control region of pIP501 (A) and a corresponding analysis of pT181 (B) are shown. The top portions of both panels show autoradiograms obtained after hybridization with labeled cognate antisense RNAs as probes (RNAIII for pIP501 and RNAI for pT181). The middle portions are autoradiograms from reprobing the same membranes with labeled sense RNAs. The bottom portions are loading controls (hybridized with a probe against 5S rRNA). (A) pGTR6 carries an antisense RNA promoter down-mutation. Lanes none, no antisense RNA donor plasmid present; lanes pUC19, empty vector; lanes pUCpIII, pUC19 derivative providing RNAIII in trans. F and T, positions of full-length (terminated at the rrnB terminator) and prematurely terminated sense RNA (terminated at the attenuator), respectively. T*, processed form of T (see Results). (B) Both pGTR181/10 and pGTR181/35 carry antisense RNA promoter down-mutations. Lanes pMG25, insertless vector; lanes pMGI, pMG25 derivative providing RNAI in trans. Cultures were treated with IPTG for induction of antisense RNA transcription (+) or were not treated with IPTG (−) (see Materials and Methods). RNAI*, RNAI**, RNAIII*, and RNAIII** are discussed in the text. The band corresponding to F2 has its 3′ end at the 5′ side of the rrnB sequence. M, pBR322 MspI marker.

FIG. 3.

Antisense RNA-dependent attenuation in a single-round E. coli transcription system. The protocol for the single-round transcription assay is described in Materials and Methods. Autoradiograms for pIP501 (A) and pT181 (B) are shown. In all assays, transcription was terminated at 10 min. Where indicated, increasing concentrations of unlabeled homologous antisense RNA were included in the attenuation assay at concentrations between 4 × 10⁻⁸ and 8 × 10⁻⁷ M. Lanes RNAI (A) and RNAIII (B), incubations in which heterologous (unlabeled) antisense RNA at ∼8 × 10⁻⁷ M was included; lanes control, incubations in the absence of antisense RNA; lanes M, size markers. F and T, positions of full-length (runoff at the end of the template fragment) and attenuated sense RNAs, respectively.