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In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3'UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3'UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The conserved Sm and Sm-like proteins are involved in different aspects of RNA metabolism. Here, we explored the interactome of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso) to shed light on their physiological function(s). Both, SmAP1 and SmAP2 co-purified with several proteins involved in RNA-processing/modification, translation and protein turnover as well as with components of the exosome involved in 3΄ to 5΄ degradation of RNA. In follow-up studies a direct interaction with the poly(A) binding and accessory exosomal subunit DnaG was demonstrated. Moreover, elevated levels of both SmAPs resulted in increased abundance of the soluble exosome fraction, suggesting that they affect the subcellular localization of the exosome in the cell. The increased solubility of the exosome was accompanied by augmented levels of RNAs with A-rich tails that were further characterized using RNASeq. Hence, the observation that the Sso SmAPs impact on the activity of the exosome revealed a hitherto unrecognized function of SmAPs in archaea., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5'-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5'-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.

In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (Hfq(Ec)) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq-mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of Hfq(Ec) with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA(34)). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of Hfq(Ec) in complex with DsrA(34) revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA(34) is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq.

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, sigma(S), involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind.

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 gamma- and beta-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.

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the beta-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

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.

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.

The first complete nucleotide sequences of two lytic Staphylococcus aureus double stranded DNA phages, 44AHJD (16784 bp) and P68 (18227 bp), are reported. Both are small isometric phages, with short, non-contractile tails and a pre-neck appendage. Based on their morphology, their genome size, the similarity of the encoded gene products, the type of infection and on the possession of a type B DNA polymerase, 44AHJD and P68 are allocated to the order Caudovirales, family Podoviridae, genus 'phi29-like phages'. The genome of 44AHJD differs from that of P68 by a deletion spanning nucleotides 10091 to 11531 of the P68 genome. The electrophoretic analysis of the terminal DNA fragments of P68 DNA and P68 DNA protein complex suggested the presence of a terminal protein at either DNA end. In contrast to the lysis cassette of the phi29-like phages, which is located at the end of the late operon, the lysis cassette of 44AHJD and P68 is located within the structural genes.

Aims: To evaluate the ability of a filamentous phage encoding lethal proteins to kill bacteria without host-cell lysis., Methods and Results: Bacterial survival was determined after infection of a growing Escherichia coli culture with phage M13 encoding either the restriction endonuclease BglII gene or modified phage lambda S holin genes. The genetically engineered phage exerted a high killing efficiency while leaving the cells structurally intact. When compared with a lytic phage, the release of endotoxin was minimized after infection with the genetically modified phages., Conclusions: Genetically engineered phage can be used for efficient killing, concomitantly minimizing endotoxin release., Significance and Impact of the Study: This feasibility study provides a possible strategy for the use of genetically engineered phage as bactericidal agents by optimizing the advantages and minimizing potential risks such as release of pyrogenic cell wall components.

Leaderless mRNAs beginning with a 5'-terminal start codon occur in all biological systems. In this work, we have studied the comparative translational efficiency of leaderless and leadered mRNAs as a function of temperature by in vitro translation competition assays with Escherichia coli extracts. At low temperature (25 degrees C) leaderless mRNAs were found to be translated comparatively better than mRNAs containing an internal canonical ribosome binding site, whereas at high temperature (42 degrees C) the translational efficiency of canonical mRNAs is by far superior to that of leaderless mRNA. The inverse correlation between temperature and translational efficiency characteristic for the two mRNA classes was attributed to structural features of the mRNA(s) and to the reduced stability of the translation initiation complex formed at a 5'-terminal start codon at elevated temperature.

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the E phi S fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the E phi S fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.

The phage Lambda PRM promoter-derived cI mRNA and phage P2 gene V mRNA are transcribed beginning with the A residue of the AUG start codon. Using lacZ fusion analysis we have assessed the effects of alterations in the immediate downstream coding region on the translational efficiency of these mRNAs. Mutations, including deletions of the putative downstream box of either cI or gene V mRNAs, showed no significant reduction in expression of the different lacZ fusions. Primer extension inhibition analysis suggests a role of ribosomal protein S1 in cI mRNA recognition.

During the lytic cycle of most bacteriophages, a phage-encoded peptidoglycan-degrading activity is elaborated. At least four entirely distinct types of enzymes fulfill this role and are given the generic name 'endolysin'. Endolysins characterized to date are synthesized without a signal sequence and thus accumulate fully folded and active in the cytosol during the vegetative phase. Small membrane proteins are required in order for endolysins to gain access to the peptidoglycan. Because the available data suggest that the membrane lesion formed by these proteins is stable and non-specific, these proteins have been given the designation 'holins' ('hole'-formers). Analysis of the primary sequence suggests a simple membrane topology with two or more membrane-spanning helical domains and a highly charged, hydrophilic C-terminus. Comparison of the sequences of holins from phages of Gram-negative hosts suggests there are at least two major holin groups. Putative holin genes have also been found in bacteriophages of Gram-positive bacteria. Altogether, in phages of Eubacteria, 11 or more unrelated gene families which share the functional and structural characteristics of holins have been identified. Genetic and physiological analysis suggest that holins are primarily regulated at the level of function. Holin function is modulated in some cases by a second protein encoded by the holin gene. The primary regulation of holin function, however, appears to be intrinsic to the holin structure itself, since a missense allele of the S holin gene of phage lambda has been found which abolishes the normal delay that allows the vegetative phase to generate a useful number of progeny.

Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.