The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a b-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.Chaperonins are ATP-dependent molecular machines that are able to bind misfolded protein substrates and promote their proper folding (1). Protein misfolding is the main mechanism involved in a large number of genetic diseases caused by missense mutations, giving rise to the concept of "conformational diseases " (2, 3). The role of chaperones in the pathogenesis of conformational diseases has been recently reviewed (4). A deficiency of the hepatic enzyme alanine glyoxylate aminotransferase (AGXT, 2 also known as AGT) is responsible for the rare hereditary disease primary hyperoxaluria type 1 (PH1, Online Mendelian Inheritance in Man (OMIM) 259900). Insufficient AGXT activity leads to increased conversion of the substrate glyoxylate to the toxic ion oxalate, which subsequently accumulates as insoluble calcium salt in the kidney. In the later stages of the disease, calcium oxalate deposition becomes widespread and life-threatening unless liver and kidney transplantation is performed (5). Most of the PH1 alleles detected in the Canary Islands patients carry the I244T mutation together with the polymorphisms P11L and I340M. These amino acid changes result in a misfolded protein (AGXT-LTM) that undergoes stable interaction with molecular chaperones and aggregation (6). A large number of the pathogenic variants of this protein are the result of missense mutations that are likely to result in misfolded proteins (7). Thus, the study of these folding intermediates and the effect of the point mutations on the structure of the protein are central to elucidate the molecular basis responsible for the PH1 disease. The bacterial chaperonin-cochaperonin GroEL-GroES system is well characterized both at the biochemical and structural level (8, 9). Gro...

We experimentally verify the design of a low-cost scanner that performs dynamic whole-eye scans without requiring any moving component. We present beam profiles and OCT images in an ex vivo rabbit eye.