Structure of the Bacteriophage PhiKZ Non-virion RNA Polymerase Transcribing from its Promoter p119L.

Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people worldwide each year. ΦKZ infection is independent of the host transcriptional apparatus; the virus forms a "nucleus", producing a proteinaceous barrier around the ΦKZ genome that excludes the host immune systems. It expresses its own non-canonical multi-subunit non-virion RNA polymerase (nvRNAP), which is imported into its "nucleus" to transcribe viral genes. The ΦKZ nvRNAP is formed by four polypeptides representing homologues of the eubacterial ß/ß' subunits, and a fifth that is likely to have evolved from an ancestral homologue to σ-factor. We have resolved the structure of the ΦKZ nvRNAP initiating transcription from its cognate promoter, p119L, including previously disordered regions. Our results shed light on the similarities and differences between ΦKZ nvRNAP mechanisms of transcription and those of canonical eubacterial RNAPs and the related non-canonical nvRNAP of bacteriophage AR9.

Architecture of the Tuberous Sclerosis Protein Complex.

The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.

Nutrient Signaling and Lysosome Positioning Crosstalk Through a Multifunctional Protein, Folliculin.

FLCN was identified as the gene responsible for Birt-Hogg-Dubé (BHD) syndrome, a hereditary syndrome associated with the appearance of familiar renal oncocytomas. Most mutations affecting FLCN result in the truncation of the protein, and therefore loss of its associated functions, as typical for a tumor suppressor. FLCN encodes the protein folliculin (FLCN), which is involved in numerous biological processes; mutations affecting this protein thus lead to different phenotypes depending on the cellular context. FLCN forms complexes with two large interacting proteins, FNIP1 and FNIP2. Structural studies have shown that both FLCN and FNIPs contain longin and differentially expressed in normal versus neoplastic cells (DENN) domains, typically involved in the regulation of small GTPases. Accordingly, functional studies show that FLCN regulates both the Rag and the Rab GTPases depending on nutrient availability, which are respectively involved in the mTORC1 pathway and lysosomal positioning. Although recent structural studies shed light on the precise mechanism by which FLCN regulates the Rag GTPases, which in turn regulate mTORC1, how FLCN regulates membrane trafficking through the Rab GTPases or the significance of the intriguing FLCN-FNIP-AMPK complex formation are questions that still remain unanswered. We discuss the recent progress in our understanding of FLCN regulation of both growth signaling and lysosomal positioning, as well as future approaches to establish detailed mechanisms to explain the disparate phenotypes caused by the loss of FLCN function and the development of BHD-associated and other tumors.