Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site.

The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.RNA chaperone | regulation of translation | RNA degradation | prokaryotes H fq is an abundant and widely conserved RNA-binding protein in bacteria and a major player in the RNA-based regulation of gene expression, such as in the adaptive response to cell stress or in the induction of virulence (1, 2).Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8). The most prominent function of Hfq, however, is its interaction with small regulatory RNAs (sRNAs) that act in trans and that are differentially expressed under various metabolic and environmental conditions (9, 10). They globally regulate gene expression via base-pairing to frequently entire sets of partially complementary mRNAs (11,12). Hfq stabilizes sRNAs in the absence of their targets (13). Hfq was also found to facilitate base-pairing to the mRNAs and help trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (11,14). Despite their structural diversity, the recognition of many sRNAs by Hfq is highly specific and even works across species barriers (15). It is an intriguing question how this selectivity is achieved.Crystal structures reveal that bacterial Hfq adopts an (L)Sm fold and forms homohexameric rings, whereas related (L)Sm proteins [Sm proteins and Sm-like (LSm) proteins] in archaea and eukaryotes are found to form homomeric or heteromeric heptamers, respectively (16). Two distinct RNA-binding sites have been described on opposite f...

The bacterial Sm-like protein Hfq is a central player in the control of bacterial gene expression. Hfq forms complexes with small regulatory RNAs (sRNAs) that use complementary "seed" sequences to target specific mRNAs. Hfq forms hexameric rings, which preferably bind uridine-rich RNA 3′ ends on their proximal surface and adeninerich sequences on their distal surface. However, many reported properties of Hfq/sRNA complexes could not be explained by these RNA binding modes. Here, we use the RybB sRNA to identify the lateral surface of Hfq as a third, independent RNA binding surface. A systematic mutational analysis and competition experiments demonstrate that the lateral sites have a preference for and are sufficient to bind the sRNA "body," including the seed sequence. Furthermore, we detect significant structural rearrangements of the Hfq/sRNA complex upon mRNA target recognition that lead to a release of the seed sequence, or of the entire sRNA molecule in case of an unfavorable 3′ end. Consequently, we propose a molecular model for the Hfq/sRNA complex, where the sRNA 3′ end is anchored in the proximal site of Hfq, whereas the sRNA body, including the seed sequence, is bound by up to six of the lateral sites. In contrast to previously proposed arrangements, the presented model explains how Hfq can protect large parts of the sRNA body while still allowing a rapid recycling of sRNAs. Furthermore, our model suggests molecular mechanisms for the function of Hfq as an RNA chaperone and for the molecular events that are initiated upon mRNA target recognition. (4). Similar to its heteroheptameric homologs in eukaryotes, the Hfq ring has a socalled proximal RNA binding site, where it accommodates up to six nucleotides (one per monomer), with a preference for uridines and a discrimination against guanines (5-7). In addition to the proximal surface, the distal surface of the Hfq ring also binds RNA, with a preference for adenine-rich sequences, and covering up to 18 nucleotides-two (8) or three (9) per monomer.The regulatory sRNAs that bind Hfq are Rho-independent transcription units and consequently share a terminator stemloop structure with a stretch of single-stranded uridines at their 3′ end (10); their remaining bodies are structurally very diverse, with additional hairpins and single-stranded uridine-rich regions (11,12). Depending on the metabolic and environmental conditions, sRNAs are differentially expressed (13, 14) and regulate gene expression via base pairing, frequently to entire sets of partially complementary mRNAs (15, 16). The respective mRNA targeting (seed) sequence is usually located at the 5′ end of the sRNA (11,12).Hfq stabilizes sRNAs in the absence of their targets and facilitates base-pairing to the mRNAs (17-19); it also helps trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (2,3,16,20). Furthermore, there is a rapid exchange and competition of sRNAs for Hfq under physiological conditions, such that Hfq can be regarded...

Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box–peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.DOI: http://dx.doi.org/10.7554/eLife.07888.001

Over the past years, small non-coding RNAs (sRNAs) emerged as important modulators of gene expression in bacteria. Guided by partial sequence complementarity, these sRNAs interact with target mRNAs and eventually affect transcript stability and translation. The physiological function of sRNAs depends on the protein Hfq, which binds sRNAs in the cell and promotes the interaction with their mRNA targets. This important physiological function of Hfq as a central hub of sRNA-mediated regulation made it one of the most intensely studied proteins in bacteria. Recently, a new model for sRNA binding by Hfq has been proposed that involves the direct recognition of the sRNA 3' end and interactions of the sRNA body with the lateral RNA-binding surface of Hfq. This review summarizes the current understanding of the RNA binding properties of Hfq and its (s)RNA complexes. Moreover, the implications of the new binding model for sRNA-mediated regulation are discussed.