O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.

SUMMARY A crucial step in protein translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunits is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize for the first time the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69 and H38 that is caused by the ratcheting of the small subunit.

Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.hanges in ribosome conformation during protein synthesis are substantial, the most pronounced ones occurring during mRNA-tRNA translocation along the A (aminoacyl), P (peptidyl), and E (exit) tRNA binding sites of the ribosome, as postulated early on by Spirin (1) and Bretscher (2) and shown in recent studies by cryo-EM, X-ray crystallography, and smFRET [see (3)]. These changes go along with changes in binding configurations the ribosome forms with the tRNAs and elongation factor G (EF-G) in the process of translocation.Translocation can be broadly divided into two phases [see (4)]: during the first phase, the tRNAs move with respect to the large (50S) subunit, and in the second, the mRNA and the tRNAs affixed to it move with respect to the small (30S) subunit. After accommodation of the incoming aminoacyl-tRNA into the A/A site, and peptide transfer from the peptidyl-tRNA residing in the P/P site, the tRNAs proceed from the classical (A/A, P/P) to the hybrid (A/P, P/E) binding configuration (5): while the anticodon stem loops (ASLs) of tRNAs stay in the small subunit's A and P sites, the acceptor ends move to the large subunit's P and E sites, respectively. As EF-G binds to the complex, the ribosome undergoes a ratchet-like motion ("intersubunit rotation")-the 30S subunit rotates with respect to the 50S subunit (6). This rotation is accompanied by a movement of the L1 stalk of the 50S subunit toward the main body of the ribosome and a rotation of the head domain of the 30S subunit around its long axis (7-10). These movements separate two distinct states during the first phase of translocation, termed macrostate I (MS I) and II (MS II) (4). The fact that the conformational changes of the ribosome and classical-hybrid transitions of tRNAs occur spontaneously in a pretrans-locational (PRE) ribosome (11-16) has confirmed the view of the ribosome as a Brownian machine (17). In this view, the role of ribosomal factors is to modulate the free-energy landscape, promoting or controlling structural and kinetic routes underlying functional dynamics of translation (18,19). In the case of translocation, smFRET has provided rich detail on the way EF-G promotes and controls the reaction [e.g., (13, 14, 20-27)].Cryo-EM (11, 15) of factor-free ...