Isabella Moll

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control.

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.

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

Control of Fur synthesis by the non‐coding RNA RyhB and iron‐responsive decoding

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.

RNA chaperone activity of the Sm-like Hfq protein.

The Escherichia coli Sm‐like host factor I (Hfq) protein is thought to function in post‐transcriptional regulation by modulating the function of small regulatory RNAs. Hfq also interferes with ribosome binding on E. coli ompA messenger RNA, indicating that Hfq also interacts with mRNAs. In this study, we have used stimulation of group I intron splicing in vivo and a modified in vitro toeprinting assay to determine whether Hfq acts as an RNA chaperone. Hfq was able to rescue an RNA ‘folding trap’ in a splicing defective T4 bacteriophage td gene in vivo. Enzymatic analysis showed that Hfq affects the accessibility of the ompA start codon, as well as other bases within the ribosome‐binding site, explaining its negative effect on ribosome binding. We also show that the Hfq‐induced structural changes in ompA mRNA are maintained after proteolytic digestion of the protein, which classifies Hfq as an RNA chaperone.

Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli

The Escherichia coli Sm‐like host factor I (Hfq) is thought to play direct and indirect roles in post‐transcriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq– background. The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.

Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools

Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless λcI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsBts mutant. The analysis of the ribosomes isolated under the non‐permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non‐permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the ‘DeaD/CsdA’ DEAD‐box helicase, has been isolated as a multicopy suppressor of rpsBts mutations. Here, we show that expression of a plasmid borne deaD/csdA gene restores both S1 and S2 on the ribosome at the non‐permissive temperature in the rpsBts strain, which in turn leads to suppression of the translational defect affecting canonical mRNAs. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5′‐terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λcI and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non‐standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.

Structure of Pseudomonas aeruginosa Hfq protein.

The structure of the Hfq protein from Pseudomonas aeruginosa was determined using two different ionic conditions. In both cases the molecules formed identical hexameric rings, but some variations in the crystal packing were revealed. Hfq belongs to the family of Sm/LSm proteins, the members of which can form hexameric as well as heptameric rings. Comparative analysis of known structures of this protein family shows that the fragment of the Sm-fold responsible for oligomerization is strongly structurally conserved. In the heptameric ring, three conserved hydrogen bonds between beta-strands of adjacent molecules hold together the monomers, whereas in the hexameric rings of Hfq an additional conserved inaccessible hydrogen bond between neighbouring monomers is observed.

Functional replacement of the Escherichia coli hfq gene by the homologue of Pseudomonas aeruginosa

The 102 aa Hfq protein of Escherichia coli (Hfq(Ec)) was first described as a host factor required for phage Qbeta replication. More recently, Hfq was shown to affect the stability of several E. coli mRNAs, including ompA mRNA, where it interferes with ribosome binding, which in turn results in rapid degradation of the transcript. In contrast, Hfq is also required for efficient translation of the E. coli and Salmonella typhimurium rpoS gene, encoding the stationary sigma factor. In this study, the authors have isolated and characterized the Hfq homologue of Pseudomonas aeruginosa (Hfq(Pa)), which consists of only 82 aa. The 68 N-terminal amino acids of Hfq(Pa) show 92% identity with Hfq(Ec). Hfq(Pa) was shown to functionally replace Hfq(Ec) in terms of its requirement for phage Qbeta replication and for rpoS expression. In addition, Hfq(Pa) exerted the same negative effect on E. coli ompA mRNA expression. As judged by proteome analysis, the expression of either the plasmid-borne hfq(Pa) or the hfq(Ec) gene in an E. coli Hfq(-) RpoS(-) strain revealed no gross difference in the protein profile. Both Hfq(Ec) and Hfq(Pa) affected the synthesis of approximately 26 RpoS-independent E. coli gene products. These studies showed that the functional domain of Hfq resides within its N-terminal domain. The observation that a C-terminally truncated Hfq(Ec) lacking the last 27 aa [Hfq(Ec(75))] can also functionally replace the full-length E. coli protein lends further support to this notion.