Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

• MeSH
• elektronová kryomikroskopie MeSH
• genom virový genetika   MeSH
• molekulární chaperony metabolismus   MeSH
• molekulární modely MeSH
• proteiny vázající RNA metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA virová genetika   MeSH
• Rotavirus genetika   růst a vývoj   metabolismus   MeSH
• sbalování RNA genetika   MeSH
• virové nestrukturální proteiny metabolismus   MeSH
• zabalení virového genomu genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.

• MeSH
• Escherichia coli metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• molekulární chaperony genetika   metabolismus   MeSH
• peptidylprolylisomerasa genetika   metabolismus   MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• hydrolýza MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny - lokalizační signály genetika   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• protonmotorická síla * MeSH
• translokační kanály SEC chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH