Volume 18 Issue 11, November 2011

The microRNA-induced silencing complex (miRISC) protein TNRC6 (also called GW182) uses dispersed tryptophan-containing repeats in unstructured regions to recruit the CCR4–NOT nuclease complex leading to mRNA deadenylation and inhibition of translation initiation according to new research.

Organism-wide RNA interference (RNAi) is due to the movement of mobile RNA species throughout the organism. New genetic evidence shows that double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNAs in Caenorhabditis elegans. Interestingly, single-stranded primary and amplified secondary siRNAs do not generate mobile silencing RNAs.

Deficiencies in aprataxin, which reverses 5′-adenylate DNA adducts, can lead to the neurodegenerative disorder AOA1. Mutagenesis analyses and the crystal structure of the aprataxin ortholog from Schizosaccharomyces pombe in complex with DNA, AMP and Zn²⁺ reveal the mechanisms by which this enzyme processes DNA lesions and maintains genome integrity.

NuA4 is an essential and conserved histone acetyltransferase (HAT) complex. Using electron microscopy supported by biochemical analyses, insights are now gained into its interaction with the nucleosome core particle (NCP). These data indicate that the Epl1 subunit is essential for NCP interaction, whereas the Yng2 subunit positions the acetyltransferase complex relative to specific histone tails.

LRP5/6 is a Wnt co-receptor essential for Wnt/β-catenin signaling. X-ray crystallography and electron microscopy analyses now reveal that the LRP6 extracellular ligand-binding domain is composed of two pairs of rigid blocks connected by a short hinge. The structure of the LRP6–DKK1c complex also shows how DKK1 may potently inhibit Wnt/β-catenin signaling through its binding to these structural blocks.

The GW182 protein, which binds poly(A)-binding protein (PABP) and is part of the miRNA-induced silencing complex, effects translational repression and deadenylation of target mRNAs. New data indicate that GW182 independently interacts with the PAN2–PAN3 and CCR4–NOT deadenylase complexes and that interaction of GW182 with CCR4–NOT is PABP-independent and occurs through discrete binding sites with distinct roles in miRNA-mediated deadenylation.

The function of GW182 proteins in miRNA-mediated gene silencing has been unclear. Studies in human and fly cells now identify tryptophan-containing motifs as important repressive elements. These motifs, which are dispersed throughout GW182 proteins, function additively by recruiting deadenylase complexes to repress both poly(A)⁺ and poly(A)⁻ mRNAs, suggesting that recruitment, in addition to catalyzing deadenylation, also mediates translational repression.

Eukaryotic initiation factor eIF2 binds initiator Met-tRNA_i^Met to the P site of the 40S ribosomal subunit. Hydroxyl radical probing analyses allowed mapping of the binding of eIF2 on the ribosome and on Met-tRNA_i^Met. Interestingly, despite having structural similarities to elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the A site of 70S elongating ribosomes, eIF2 binds tRNA in a substantially different manner.

Monoclonal antibody 2F5 recognizes the membrane proximal external region (MPER) in GP41 and has broad neutralization activity against HIV-1 strains. NMR, EPR and HDX/MS analyses now show that 2F5 extracts MPER from the membrane using a scoop-like movement.

Dimerization, ligand-binding and co-confinement are all expected to contribute to erbB1 signaling, but the level of their contribution has not yet been fully appreciated. Two-color quantum-dot tracking in live cells now shows that ligand-binding (two ligands with two receptors) stabilizes erbB1 dimers, whereas actin networks influence dimer mobility and promote repeated encounters between erbB1 monomers.

Tubulin undergoes cycles of removal or addition of a C-terminal tyrosine residue to the C-terminus of α-tubulin within the α-β heterodimer. Crystal structures of tubulin tyrosine ligase (TTL), along with SAXS and functional analyses, reveal that TTL interacts with the C-terminal tail of α-tubulin and also with its longitudinal face, preventing incorporation of the α–β tubulin dimer into the microtubule lattice.

The eukaryotic proteasome is composed of a regulatory particle and a core particle. Now protein cross-linking is used to map the contacts between regulatory particle and core particle subunits, revealing that some Rpt subunits engage with the core particle in a dynamic fashion that is regulated by nucleotide binding and hydrolysis.

Archaea use a tRNA^Ile with agmatine conjugated at the C34 position to decode the AUA codon. The enzyme that catalyzes this reaction, TiaS, has been previously identified, and it is now shown that it uses a novel kinase domain to hydrolyze ATP into AMP and pyrophosphate before phosphorylating itself and the tRNA on its way to conjugating the agmatine.

TiaS catalyzes the transfer of agmatine onto the first position cytidine of the tRNAIle2 anticodon in archaea, ensuring proper translation of the matching codon. Now the crystal structures of the TiaS–tRNAIle2 complex with ATP, or with AMPCPP and agmatine, reveal a novel kinase domain and show how TiaS selects the correct tRNA while segregating the target cytidine until agmatine is bound.

The human mitochondrial transcription factor TFAM is essential for DNA packaging as well as transcription. X-ray analysis of TFAM in complex with a mitochondrial promoter reveals that TFAM induces a 180-degree bend in the DNA, which creates an optimal DNA arrangement for transcription initiation, while facilitating DNA compaction of the mitochondrial genome elsewhere.

The mitochondrial transcription factor Tfam has a role in organizing the mitochondrial genome, in addition to its transcriptional function. Structural studies of human Tfam in complex with a mitochondrial DNA promoter show that Tfam imposes a U-turn on the DNA similarly to the unrelated HU family of proteins, which play analogous architectural roles in organizing bacterial nucleoids.

Aprataxin is a DNA deadenylase that processes DNA molecules with 5'-AMP termini that result from abortive ligation reactions. The crystal structures of the Schizosaccharomyces pombe ortholog Hnt3 in its apo state, bound to DNA or DNA and AMP, provide insight into how this enzyme recognizes and processes its substrate.

During protein synthesis, mRNA and tRNAs are iteratively translocated by the ribosome, but which molecular event is rate limiting for translocation has been unknown. Kinetics analyses now reveal that disruption of the interactions between the A-site codon and the ribosome accelerates translocation, suggesting that mRNA release from the decoding center of the ribosome is the rate-limiting step.