Summary Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3′ terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Q␤ replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq − strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control trast, the mechanism(s) leading to translation of leaderless mRNA, in which the start codon is either preceded by only a few nucleotides or which starts directly with a 5¢-terminal AUG, has remained elusive. In this review, we summarize the current knowledge on translation of leaderless mRNAs in Escherichia coli and discuss their possible biological implication. Ribosomal recruitment signals on leaderless mRNAs downstream of the start codon: do they exist?The finding of a Watson and Crick complementarity between the initiation-proximal coding region (downstream box, DB) of several phage and bacterial mRNAs and bases 1469-1483 within helix 44 of 16S rRNA (anti-DB, aDB) was at the origin of the hypothesis that, similarly to the Shine-Dalgarno (SD)-anti-SD interaction, a DB-aDB pairing could enhance the translational efficiency (Sprengart et al., 1990). Soon afterwards, it was suggested that the DB could compensate for the absence of a SD sequence in leaderless mRNAs (Shean and Gottesman, 1992). However, evidence in support of the DB-aDB interaction from biochemistry, mRNA-rRNA co-variation and rRNA mutagenesis is invariably lacking. Chemical probing studies have failed to show protection of the putative DB of the leaderless lcI mRNA bound in 70S initiation complexes (Resch et al., 1996). Moreover, the 3D crystal structure of the Thermus thermophilus 30S subunit (Wimberley et al., 2000) revealed that the whole shoulder of the body of the ribosomal particle is situated between the putative DB of the mRNA and that part of helix 44 of 16S rRNA comprising the proposed aDB . This renders any model in which the start codon is placed in the ribosomal P-site whereas the adjacent DB interacts with the aDB (Sprengart and Porter, 1997) or in which the DB basepairs synergistically with the SD-anti-SD interaction (Sprengart et al., 1996) untenable. Likewise, there is no experimental support for the speculation that a DB-aDB basepairing could contribute to the initial 30S-mRNA interaction before translation initiation complex formation (Sprengart and Porter, 1997). Neither natural lcI mRNA nor a derivative of this leaderless mRNA with an optimized basepairing potential with the aDB formed a binary complex with ribosomes . This result was consistent with previous kinetic toeprint experiments (Resch et al., 1996), as well as with the findings that the aDB is not accessible to a Molecular Microbiology (2002) 43(1), 239

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.

Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5′ part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

The Fe 2 þ -dependent Fur protein serves as a negative regulator of iron uptake in bacteria. As only metallo-Fur acts as an autogeneous repressor, Fe 2 þ 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. RyhBmediated regulation of uof-fur provides the first example for indirect translational regulation by a trans-encoded ncRNA. In addition, we present evidence for an ironresponsive 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.

scientific report 284The 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.

Translation of leaderless mRNAs, lacking ribosomal recruitment signals other than the 5'-terminal AUG-initiating codon, occurs in all three domains of life. Contemporary leaderless mRNAs may therefore be viewed as molecular fossils resembling ancestral mRNAs. Here, we analyzed the phenomenon of sustained translation of a leaderless mRNA in the presence of the antibiotic kasugamycin. Unexpected from the known in vitro effects of the drug, kasugamycin induced the formation of stable approximately 61S ribosomes in vivo, which were proficient in selectively translating leaderless mRNA. 61S particles are devoid of more than six proteins of the small subunit, including the functionally important proteins S1 and S12. The lack of these proteins could be reconciled with structural changes in the 16S rRNA. These studies provide in vivo evidence for the functionality of ribosomes devoid of multiple proteins and shed light on the evolutionary history of ribosomes.