Articles in 2012

In order to locate the voltage-dependent gate in the MthK potassium channel, intracellular quaternary ammonium blockers are used for electrophysiology and crystallographic analyses. The data conclusively show that the inactivation gate is located at the selectivity filter and not at the cytoplasmic bundle crossing entrance.

A biochemical approach is used to understand how the ULK1 complex integrates signals from ATG proteins during autophagy. The interaction between ULK1-associated protein FIP200 and ATG16L1 of the ATG5 complex is revealed. This interaction is important for autophagy induced by amino acid starvation but not by glucose deprivation.

Rare or nonoptimal codons that cause ribosomes to pause have been suggested to be important determinants of cotranslational folding. A revised translational efficiency scale, which considers tRNA abundance as well as codon usage and codon-tRNA interaction, now suggests a correlation between optimal or nonoptimal codon usage and secondary structure of the nascent polypeptide.

The transcription machinery must locate specific promoter sequences among a vast excess of nonspecific DNA. Real-time single-molecule experiments with E. coli RNA polymerase, combined with theoretical calculations, suggest that facilitated diffusion does not contribute to promoter targeting at physiologically relevant protein concentrations but that instead the promoter search is dominated by three-dimensional diffusion.

Studies of the hepatitis C virus internal ribosome entry site (IRES) mechanism have focused on how IRES assembles an 80S ribosome at the start codon. Structural and functional analyses demonstrate that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause conformational changes and that the mutated domain decreases IRES activity by inhibiting ribosome translocation and, thereby, translation elongation.

The COPII cage, formed by Sec13 and Sec31, organizes other proteins into a lattice on the endoplasmic-reticulum membrane and is involved in transporting cargo from the endoplasmic reticulum to the Golgi apparatus. A combination of cryo-EM and H/D-exchange MS analyses leads to a 12-Å-resolution model of the COPII cage, yielding insight into its architecture and assembly.

A cell-based screen for intronic splicing silencers revealed ten sequence motifs that inhibited splicing in human cells and either enhanced or inhibited exon inclusion when inserted into exons. Identification of trans-acting splicing factors for each motif revealed a complex network, which suggests that cis elements function differently in distinct cellular contexts, depending on the regulatory factors present.

Dengue virus has two membrane proteins, E and M, which undergo dramatic structural changes during the life cycle of the virus. The 3.5-Å cryo-EM structure of the mature prefusion Dengue virion reveals the detailed interactions between E and M, providing insight into how conformational changes are triggered.

A comprehensive metagenomic analysis of chromatin immunoprecipitation–sequencing and microarray analysis (ChIP-seq and ChIP-chip) data from mouse embryonic stem cells provides insight into how histone gene transcription is controlled. The work reveals a complex mode of regulation, with multiple factors acting to regulate transcription of core and linker histones.

The transmembrane export apparatus regulates protein secretion through bacterial type III secretion systems. New structural data indicate that MxiA, a major component of the apparatus, assembles in a nonameric ring. This and additional structural information provide a framework for understanding how protein secretion is controlled.

Glucocorticoid receptor (GR) transactivates genes containing the response element GRE. GR can also mediate transrepression of genes by binding to the so-called negative GRE (nGRE). The interaction between GR and nGRE is now revealed by structural and functional approaches, showing that two GR monomers bind nGRE in a unique conformation and with strong negative cooperativity.

HLA-DM interacts with MHCII and promotes peptide exchange. This activity of HLA-DM is regulated by HLA-DO. The crystal structure of the HLA-DO–HLA-DM complex along with mutagenesis and kinetic analyses reveal that HLA-DO adopts a classical MHCII structure and competitively inhibits HLA-DM's activity on MHCII.

Formins regulate actin nucleation and filament elongation through their conserved FH2 domain. The formin FMNL3 induces assembly of filopodia, and now the crystal structure of its FH2 domain in complex with actin, together with functional analyses, provides insight into formin's activities.

mRNA-binding proteins have crucial regulatory functions in gene expression. A global analysis in budding yeast now uncovers 120 proteins that cross-link to mRNA, including 66 new mRNA-binding proteins. CLIP analyses of P-body components further identify sites of interaction on specific mRNAs, revealing principles by which these proteins assemble into P-body mRNPs.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and are key regulators of the post-transcriptional fate of messenger ribonucleoprotein particles (mRNPs). Two recent papers reporting on the transcriptome-wide mapping of EJC-binding sites in human cells reveal an unexpected heterogeneity of EJC distribution on mRNAs and a tight network of EJC–SR protein interactions contributing to the formation of a higher-order, compacted mRNP structure.

A newly uncovered activity of a family of Polycomb-group proteins provides insight into the mechanisms by which active genes become repressed during the transition from pluripotency to restricted cell fates as stem cells undergo lineage specification.

Three structures were recently reported for tyrosyl DNA phosphodiesterase 2 (Tdp2), a protein that protects the metazoan genome from shredding following abortive topoisomerase activity. The physiological significance of these findings is discussed here.

This report describes the outcomes of the Data Management Challenges in 3D Electron Microscopy workshop. Key topics discussed include data models, validation and raw-data archiving. The meeting participants agreed that the EMDataBank should take the lead in addressing these issues, and concrete action points were agreed upon that will have a substantial impact on the accessibility of three-dimensional EM data in biology and medicine.

Histone post-translational modifications (PTMs) can directly influence histone-DNA and histone-histone interactions, or they can be targeted by protein effectors, or histone readers. This Review outlines known readers of histone PTMs, details their mechanism of action and the functional significance of histone PTM recognition and discusses cross-talk between protein effectors and consequences of the combinatorial readout of PTMs.