Summary: It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5′‐terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome‐IF2‐fMet‐tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of lead‐erless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evo‐lutionarily conserved function of the translational apparatus.

Summary: Quorum sensing, a cell‐to‐cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans‐encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum‐sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base‐pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen‐responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen‐limited growth in P. aeruginosa biofilms.

Carbon Catabolite repression (CCR) allows a fast adaptation of Bacteria to changing nutrient supplies. The Pseudomonas aeruginosa (PAO1) catabolite repression control protein (Crc) was deemed to act as a translational regulator, repressing functions involved in uptake and utilization of carbon sources. However, Crc of PAO1 was recently shown to be devoid of RNA binding activity. In this study the RNA chaperone Hfq was identified as the principle post-transcriptional regulator of CCR in PAO1. Hfq is shown to bind to A-rich sequences within the ribosome binding site of the model mRNA amiE, and to repress translation in vitro and in vivo. We further report that Crc plays an unknown ancillary role, as full-fledged repression of amiE and other CCR-regulated mRNAs in vivo required its presence. Moreover, we show that the regulatory RNA CrcZ, transcription of which is augmented when CCR is alleviated, binds to Hfq with high affinity. This study on CCR in PAO1 revealed a novel concept for Hfq function, wherein the regulatory RNA CrcZ acts as a decoy to abrogate Hfq-mediated translational repression of catabolic genes and thus highlights the central role of RNA based regulation in CCR of PAO1. Carbon assimilation in Bacteria is governed by a mechanism known as carbon catabolite repression (CCR). In contrast to several other bacterial clades CCR in Pseudomonas species appears to be primarily regulated at the post-transcriptional level. In this study, we have identified the RNA chaperone Hfq as the principle post-transcriptional regulator of CCR in P. aeruginosa (PAO1). Hfq is shown to act as a translational regulator and to prevent ribosome loading through binding to A-rich sequences within the ribosome binding site of mRNAs, which encode enzymes involved in carbon utilization. It has been previously shown that the synthesis of the RNA CrcZ is augmented in the presence of non-preferred carbon sources. Here, we show that the CrcZ RNA binds to and sequesters Hfq, which in turn abrogates Hfq-mediated translational repression of mRNAs, the encoded functions of which are required for the breakdown of non-preferred carbon sources. This novel mechanistic twist on Hfq function not only highlights the central role of RNA based regulation in CCR of PAO1 but also broadens the view of Hfq-mediated post-transcriptional mechanisms.

The Fe2+‐dependent Fur protein serves as a negative regulator of iron uptake in bacteria. As only metallo‐Fur acts as an autogeneous repressor, Fe2+scarcity would direct fur expression when continued supply is not obviously required. We show that in Escherichia coli post‐transcriptional regulatory mechanisms ensure that Fur synthesis remains steady in iron limitation. Our studies revealed that fur translation is coupled to that of an upstream open reading frame ( uof ), translation of which is downregulated by the non‐coding RNA (ncRNA) RyhB. As RyhB transcription is negatively controlled by metallo‐Fur, iron depletion creates a negative feedback loop. RyhB‐mediated regulation of uof‐fur provides the first example for indirect translational regulation by a trans ‐encoded ncRNA. In addition, we present evidence for an iron‐responsive decoding mechanism of the uof‐fur entity. It could serve as a backup mechanism of the RyhB circuitry, and represents the first link between iron availability and synthesis of an iron‐containing protein.

The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans -encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the γ- and β-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq ₆₅ ), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq ₆₅ retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.