Architectural principles for Hfq/Crc-mediated regulation of gene expression.
ABSTRACT: In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.
Project description:In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN ? factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ?crcZ?crcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases.
Project description:Hfq is an RNA chaperone and an important post-transcriptional regulator in bacteria. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), we show that Hfq associates with hundreds of different regions of the Pseudomonas aeruginosa chromosome. These associations are abolished when transcription is inhibited, indicating that they reflect Hfq binding to transcripts during their synthesis. Analogous ChIP-seq analyses with the post-transcriptional regulator Crc reveal that it associates with many of the same nascent transcripts as Hfq, an activity we show is Hfq dependent. Our findings indicate that Hfq binds many transcripts co-transcriptionally in P. aeruginosa, often in concert with Crc, and uncover direct regulatory targets of these proteins. They also highlight a general approach for studying the interactions of RNA-binding proteins with nascent transcripts in bacteria. The binding of post-transcriptional regulators to nascent mRNAs may represent a prevalent means of controlling translation in bacteria where transcription and translation are coupled.
Project description:Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.
Project description:Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H+-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.
Project description:Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes. Overall design: Total RNA from wild-ype and three mutants of P. putida KT2440 was deep-sequenced and gene expressions were quantified. Differential expression of bacterial genes for each mutant against wild type was determined.
Project description: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.
Project description:Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent ?-sheets within individual protomers to assemble into a typical toroidal hexameric ring. A C-terminal flexible domain that encompasses approximately one-third of the protein seems intrinsically unstructured. RNA-binding function of Hfq mainly lies within its N-terminal core, whereas the function of the flexible domain remains controversial and largely unknown. In the present study, we demonstrate that the Hfq-C-terminal region (CTR) has an intrinsic property to self-assemble into long amyloid-like fibrillar structures in vitro. We show that normal localization of Hfq within membrane-associated coiled structures in vivo requires this C-terminal domain. This finding establishes for the first time a function for the hitherto puzzling CTR, with a plausible central role in RNA transactions.
Project description:Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.
Project description:Small RNAs post-transcriptionally regulate many processes in bacteria. Base-pairing of sRNAs near ribosome-binding sites in mRNAs inhibits translation, often requiring the RNA chaperone Hfq. In the canonical model, Hfq simultaneously binds sRNAs and mRNA targets to accelerate pairing. Here, we show that the Escherichia coli sRNAs OmrA and OmrB inhibit translation of the diguanylate cyclase DgcM (previously: YdaM), a player in biofilm regulation. In OmrA/B repression of dgcM, Hfq is not required as an RNA interaction platform, but rather unfolds an inhibitory RNA structure that impedes OmrA/B binding. This restructuring involves distal face binding of Hfq and is supported by RNA structure mapping. A corresponding mutant protein cannot support inhibition in vitro and in vivo; proximal and rim mutations have negligible effects. Strikingly, OmrA/B-dependent translational inhibition in vitro is restored, in complete absence of Hfq, by a deoxyoligoribonucleotide that base-pairs to the biochemically mapped Hfq site in dgcM mRNA We suggest that Hfq-dependent RNA structure remodeling can promote sRNA access, which represents a mechanism distinct from an interaction platform model.
Project description:Carbapenems are often the antibiotics of choice to combat life threatening infections caused by the opportunistic human pathogen Pseudomonas aeruginosa. The outer membrane porins OprD and OpdP serve as entry ports for carbapenems. Here, we report that the RNA chaperone Hfq governs post-transcriptional regulation of the oprD and opdP genes in a distinctive manner. Hfq together with the recently described small regulatory RNAs (sRNAs) ErsA and Sr0161 is shown to mediate translational repression of oprD, whereas opdP appears not to be regulated by sRNAs. At variance, our data indicate that opdP is translationally repressed by a regulatory complex consisting of Hfq and the catabolite repression protein Crc, an assembly known to be key to carbon catabolite repression in P. aeruginosa. The regulatory RNA CrcZ, which is up-regulated during growth of P. aeruginosa on less preferred carbon sources, is known to sequester Hfq, which relieves Hfq-mediated translational repression of genes. The differential carbapenem susceptibility during growth on different carbon sources can thus be understood in light of Hfq-dependent oprD/opdP regulation and of the antagonizing function of the CrcZ RNA on Hfq regulatory complexes.