Molecular basis of tRNA recognition by the Elongator complex

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo–electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer

Mechanism of glycogen synthase inactivation and interaction with glycogenin

Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 Å resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite “arginine cradle”. Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic “spike” region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases

Etudes structure-fonction de complexes du ribosome humain

Ribosomes comprise the translational machinery engaged in synthesizing proteins. The architecture and translation regulation of eukaryotic especially, human ribosomes, has been an enigma for a long time. I established a protocol for purifying homogenous ribosomes from HeLa cells which can be used for structural as well as biochemical analysis. Using these ribosomes, I obtained plate-like crystals of 80S diffracting to low resolution. A cryo electron microscopy analysis of these ribosomes yielded 5 Å resolution structure with secondary structures of rRNA and protein clearly visible. Furthermore, these ribosomes, along with the eukaryotic release factors (eRF1 and eRF3) purified by over-expression in bacteria, formed the basis for translation termination studies using cryo electron microscopy. Simultaneously, eRF1-eRF3 protein complex was explored by X-ray crystallography revealing new interactions. Together, this work paves the way for the analysis of functional ribosome complexes.L’architecture et la régulation de la traduction eucaryote fut pendant longtemps un mystère pour les biologistes. Je présente ici un protocole détaillé pour purifier de manière homogène des ribosomes à partir de cellules HeLa, pour des études biochimiques mais également structurales. En utilisant ces ribosomes, j’ai obtenu des cristaux diffractant à faible résolution, pouvant être utilisés pour de futurs travaux. Une analyse par cryo-microscopie électronique (CME) a abouti à une structure à 5 A de résolution, permettant la construction d’un modèle. De plus, les facteurs eRF1 et eRF3 ont permis des premières études de la terminaison de la traduction par CME. Ces protéines en complexe ont également été étudiées par cristallographie aux rayons-X, montrant des interactions jusqu’alors jamais observées. L’ensemble de ce travail fournit des résultats importants pour la préparation et la description de la structure du ribosome humain, pavant la voie vers l’analyse de complexes fonctionnels

男子體操競賽簡介

<div><p>The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.</p></div

NCp7 cosediments with 80S ribosomes.

<p>(A) Sucrose gradient fractionation profile of purified 80S ribosomes (0.9 μM) incubated with NCp7 (13.3μM). Ribosome/NCp7 ratio was about 1/15. The peak fractions (8–9) were precipitated and further analyzed by western blot. (B) Western blot of fractions 8–9 from sucrose gradient fractionations performed with only NCp7 peptide (lanes 1); only 80S ribosomes (lanes 2); and 80S ribosomes and NCp7, together (lanes 3). NCp7 and ribosomal proteins were detected with polyclonal NCp7 and RpS7 antibodies, and monoclonal RpL26 antibodies. As a control, 90 nM of NCp7 peptide was loaded (4).</p

FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.

<p>FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.</p

NCp7-eGFP dynamics in HeLa cells monitored by RICS.

<p>(A) A series of confocal images of eGFP and NCp7-eGFP expressing cells was acquired. A 128x128 pixel region was analyzed by calculating the two-dimensional spatial autocorrelation function represented as a spatial correlation surface (B) that was fitted by a 3D diffusion model (C), revealing the values of the diffusion coefficients and the number of diffusing molecules.</p

FCS measurements in eGFP and NCp7-eGFP expressing HeLa cells.

<p>(A) Experimental autocorrelation function (blue) of NCp7-eGFP in HeLa cells fitted with a model for free (green) and anomalous (red) 3D diffusion. The residuals indicate that a better fit was obtained with the anomalous diffusion model. (B) Comparison of autocorrelation curves for eGFP and NCp7-eGFP diffusion in the cytoplasm of HeLa cells. Fits (solid lines) were performed with the anomalous diffusion model. (C) Histogram of the brightness analysis for eGFP and NCp7-eGFP (N = 16).</p

Intracellular distribution of NCp7-eGFP.

<p>(A) Amino acid sequence of NCp7. Confocal images of HeLa cells expressing transiently eGFP (B) or NCp7-eGFP (C, D). Comparison with the localization of DNA labeled by 1.6 μM Hoechst 33342, (C) and RNA labeled by 1 μM Pyronin Y. The cyan color of the merge panel in (C) indicates colocalization of NCp7 with DNA in the nucleus. The nearly uniform yellow color of the merge panel in (D) indicates a strong colocalization of NCp7 with RNA all over the cell.</p

Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.

<p>The D and α values are given as means +/- SD for 800 correlation curves in 16 cells (FCS) and 40 measurements in 10 cells (RICS).</p><p>Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.</p