Supramolecular organization of Hfq-like proteins.

Bacterial Hfq proteins are structural homologs of archaeal and eukaryotic Sm/Lsm proteins, which are characterized by a 5-stranded ß-sheet and an N-terminal α-helix. Previously, it was shown that archaeal Lsm proteins (SmAP) could produce long fibrils spontaneously, in contrast to the Hfq from Escherichia coli that could form similar fibrils only after special treatment. The organization of these fibrils is significantly different, but the reason for the dissimilarity has not been found. In the present work, we studied the process of fibril formation by bacterial protein Hfq from Pseudomonas aeruginosa and archaeal protein SmAP from Methanococcus jannaschii. Both proteins have high homology with E. coli Hfq. We found that Hfq from P. aeruginosa could form fibrils after substitutions in the conserved Sm2 motif only. SmAP from M. jannaschii, like other archaeal Lsm proteins, form fibrils spontaneously. Despite differences in the fibril formation conditions, the architecture of both was similar to that described for E. coli Hfq. Therefore, universal nature of fibril architecture formed by Hfq proteins is suggested.

Architectural principles for Hfq/Crc-mediated regulation of gene expression.

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.

Sm-like protein Hfq: location of the ATP-binding site and the effect of ATP on Hfq-- RNA complexes.

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq-RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins.

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins.

Hfq is a nucleic acid-binding protein that functions as a global regulator of gene expression by virtue of its interactions with several small, non-coding RNA species. Originally identified as an Escherichia coli host factor required for RNA phage Qbeta replication, Hfq is now known to post-transcriptionally regulate bacterial gene expression by modulating both mRNA stability and translational activity. Recently shown to be a member of the diverse Sm protein family, Hfq adopts the OB-like fold typical of other Sm and Sm-like (Lsm) proteins, and also assembles into toroidal homo-oligomers that bind single-stranded RNA. Similarities between the structures, functions, and evolution of Sm/Lsm proteins and Hfq are continually being discovered, and we now report an additional, unexpected biophysical property that is shared by Hfq and other Sm proteins: E.coli Hfq polymerizes into well-ordered fibres whose morphologies closely resemble those found for Sm-like archaeal proteins (SmAPs). However, the hierarchical assembly of these fibres is dissimilar: whereas SmAPs polymerize into polar tubes (and striated bundles of such tubes) by head-to-tail stacking of individual homo-heptamers, helical Hfq fibres are formed by cylindrical slab-like layers that consist of 36 subunits arranged as a hexamer of Hfq homo-hexamers (i.e. protofilaments in a 6 x 6 arrangement). The different fibrillar ultrastructures formed by Hfq and SmAP are presented and examined herein, with the overall goal of elucidating another similarity amongst the diverse members of the Sm protein family.

Cellular electron microscopy imaging reveals the localization of the Hfq protein close to the bacterial membrane.

BACKGROUND: Hfq is a bacterial protein involved in several aspects of nucleic acid transactions, but one of its best-characterized functions is to affect the post-transcriptional regulation of mRNA by virtue of its interactions with stress-related small regulatory (sRNA). METHODOLOGY AND PRINCIPAL FINDING: By using cellular imaging based on the metallothionein clonable tag for electron microscopy, we demonstrate here that in addition to its localization in the cytoplasm and in the nucleoid, a significant amount of Hfq protein is located at the cell periphery. Simultaneous immunogold detection of specific markers strongly suggests that peripheral Hfq is close to the bacterial membrane. Because sRNAs regulate the synthesis of several membrane proteins, our result implies that the sRNA- and Hfq-dependent translational regulation of these proteins takes place in the cytoplasmic region underlying the membrane. CONCLUSIONS: This finding supports the proposal that RNA processing and translational machineries dedicated to membrane protein translation may often be located in close proximity to the membrane of the bacterial cell.