The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus ( Aae ), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P 1 and P 6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U 6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.
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Polyphosphate drives bacterial heterochromatin formation
Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage–induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.
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- Award ID(s):
- 1941966
- PAR ID:
- 10377127
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 7
- Issue:
- 52
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA∙∙∙RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways—including stress response, quorum sensing, and biofilm formation—has motivated genetic and “RNAomic” studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq∙∙∙RNA interactions (in vitro). Indeed, crystallographic and biophysical studies first established Hfq as a member of the phylogenetically conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq·RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.abk0233
