Structure-function analysis of protein complexes regulating Rag signalling within the mTOR pathway and structural studies of the ΦKZ non-virion RNA polymerase

FLCN mutations are associated with Birt-Hogg-Dubé (BHD) syndrome, a hereditary syndrome which, ultimately, results in kidney cancer. FLCN forms complexes with two folliculin interacting proteins (FNIPs). The FLCN complex is a GAP for RagC/D, therefore, being a positive modulator of mTORC1. The double nucleotide-bound setting, with the Rag GTPase heterodimer being able to simultaneously bind to both GTP and GDP, is a unique feature to the Rags amongst other GTPases, which suggests that the FLCN-GAP mechanism activity towards RagC/D is likely to be novel. We have purified the Spodoptera litura FLCN-FNIP2 complex and the RagA-RagC complex. Using cryo-EM, we have provided a structural 3D model for the S.litura FLCN-FNIP2 complex. Comparison of our 3D reconstruction to that of the human FLCN-FNIP2 complex shows that the architecture and domain organisation of the FLCN-FNIP complex is conserved in S.litura and that the complex retains the same conformation in the absence of the Rag GTPase heterodimer. Bacteriophage ΦKZ is one of a few exceptional bacterial viruses to encode two multisubunit RNAPs able to transcribe all genes needed throughout the infection cycle. Previous biochemical studies have demonstrated that the ΦKZ non-virion RNAP is a 5-subunit complex able to initiate transcription from specific promoters. The complex is formed by four β/β′-like subunits and a fifth subunit with no homology to any other protein with known function but crucial for the assembly of a functional enzyme, promoter recognition, and transcription initiation. Here, we have determined for the first time the ΦKZ non-virion RNAP structure at 3.3 ˚A resolution with and without DNA using cryo-EM. The structures provide first insights into the organisation of the four split β/β’-like subunits and the location of gp68 within the complex. Moreover, structural comparisons show that gp68 is structurally similar and is positioned at the same location as bacterial σ factors.Open Acces