Anna Brandi

A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450u2009nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80u2009nt at its 5′-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense–antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem–loop motifs of the antisense region.

Sequence-specific Recognition of DNA by the C-terminal Domain of Nucleoid-associated Protein H-NS

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage λ CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::λCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::λCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr109, Arg113, Thr114, and Ala116) belonging to the same protein loop and Glu101. The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.

Translation initiation at the root of the cold-shock translational bias

Research carried out in the last two decades has shown that all living organisms, from bacteria to mammals, have evolved mechanisms to cope with the effects caused by a sudden temperature downshift (cold-shock). Following cold-stress, the mesophilic bacterium Escherichia coli enters an acclimation phase during which cell growth stops for 3–6 hours, while bulk gene expression is drastically reduced, and a set of at least 26 well characterized cold-shock genes is selectively and transiently expressed (Yamanaka, 1999; Gualerzi et al., 2003). The proteins synthesized during the cold-acclimation phase are somewhat artificially classified into early and late cold-shock proteins, and likewise early and late cold-adapted proteins accumulate in the lag phase that precedes the resumption of cell division and growth at low temperature (Figure 1A). Overall, the present perception is that the main purposes of the proteins synthesized during cold adaptation and in cold-adapted cells are: (i) to deal with unfavorable secondary structures of nucleic acids induced/stabilized by the cold, which are expected to hinder basic functions such as transcription, ribosome assembly, and translation; (ii) to oppose the cold-shock-induced decrease in membrane fluidity; (iii) to accumulate sugars which are protective against low temperature, such as trehalose; (iv) to assist protein folding at low temperatures (Graumann and Marahiel, 1998; Phadtare et al., 1999; Gualerzi et al., 2003; Weber and Marahiel, 2003).

Development of a graphene oxide-based assay for the sequence-specific detection of double-stranded DNA molecules

Graphene oxide (GO) is a promising material for the development of cost-effective detection systems. In this work, we have devised a simple and rapid GO-based method for the sequence-specific identification of DNA molecules generated by PCR amplification. The csp genes of Escherichia coli, which share a high degree of sequence identity, were selected as paradigm DNA templates. All tested csp genes were amplified with unlabelled primers, which can be rapidly removed at the end of the PCR taking advantage of the preferential binding to GO of single-stranded versus duplex DNA molecules. The amplified DNAs (targets) were heat-denatured and hybridized to a fluorescently-labelled single strand oligonucleotide (probe), which recognizes a region of the target DNAs displaying sequence variability. This interaction is extremely specific, taking place with high efficiency only when target and probe show perfect or near perfect matching. Upon GO addition, the unbound fraction of the probe was captured and its fluorescence quenched by the GO’s molecular properties. On the other hand, the probe-target complexes remained in solution and emitted a fluorescent signal whose intensity was related to their degree of complementarity.