Brief Communications in 2011

Recent work has indicated that the Escherichia coli replisome contains three DNA polymerases that are used to replicate two parental strands. A single-molecule approach is now used to compare replisomes reconstituted with two or three polymerases, revealing that the presence of a third polymerase ensures higher processivity overall and more efficient replication of the lagging strand.

The surface glycoprotein of the distinct ebolavirus responsible for the largest outbreak yet described (Sudan Gulu) has been crystallized in complex with a novel, neutralizing antibody. The crystal structure and accompanying in vitro and in vivo experiments demonstrate that the antibody functions after virus entry and illustrates a key hotspot for ebolavirus neutralization.

The Fanconi anemia (FA) DNA-repair pathway is important in processing of DNA interstrand cross-links and in resistance to exogenously added aldehyde. Genetic analyses now reveal the synthetic lethality of deficiencies in the FA pathway and formaldehyde catabolism, indicting that this pathway repairs lesions caused by endogenous formaldehyde.

The FET family proteins FUS, EWSR1 and TAF15 are RNA-binding proteins with diverse nuclear functions. PAR-CLIP analyses now reveal the genome-wide RNA targets of all three human FET proteins and of two FUS mutants that cause amyotrophic lateral sclerosis. Although the RNA-binding properties of the mutants remain unchanged, the spectrum of RNA targets is altered because of the changed subcellular localization of the mutants.

Common fragile sites (CFSs) can drive genomic instability. The basis for their fragile nature is not clear, but lymphocyte CFSs have been mapped to regions with low replication initiation events and late replication completion. These features are now used to rapidly identify CFSs in different fibroblast cells.

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.

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.

The online game Foldit invites players to solve problems involving protein structure prediction. Now Foldit players have been recruited to work on a modeling problem and ultimately solve the crystal structure of a retroviral protease that had resisted previous determination.

Inositol 1,4,5-triphosphate (InsP₃) receptors are ligand-activated calcium channels in the endoplasmic reticulum membrane, and they are responsible for the cytoplasmic Ca²⁺ efflux that triggers many cellular processes. The crystal structures of the ligand-binding domain of rat type I InsP3R in its apo and ligand-bound form reveal the conformational changes that ultimately control channel gating.

RING E3 ligases mediate transfer of ubiquitin-like proteins from an E2 ligase to a substrate, but how this occurs is a long-standing mystery. Docking E2-RING structures onto a new crystal structure of the C-terminal domain of the E3-RING CUL1 in complex with the RBX1 RING protein now shows how a conformational change in RBX1 allows for the transfer by closing a gap between CUL1 and the E2.

Eisosomes are essential for plasma membrane organization in yeast. Structural, biochemical and cell biological studies now reveal that the main eisosome components are part of the conserved BAR-domain family of proteins, which bind and shape membranes.

Ash2L is part of a complex involved in histone H3 lysine 4 trimethylation, linked to active transcription. Ash2L is now found to contain a helix-wing-helix DNA binding domain that is needed for targeting and H3K4 trimethylation at the β-globin locus control region in vivo.

Cooperativity is a universal property of biological macromolecules that is widely exploited in their regulation and function. Simulations and single-molecule FRET studies demonstrate that molecular heterogeneity lowers the cooperativity of ligand binding, suggesting that it is crucial to determine the cooperativity of individual molecules and the extent of heterogeneity in order to derive accurate molecular models.

Deg1 is an HtrA protease that participates in the turnover of the photosynthetic proteins in chloroplasts. Now the crystal structure of Deg1, along with functional work, reveals that Deg1 oligomerization and activation occur in response to acidic pH, which should be encountered in the thylakoid lumen when exposed to light.

An intein controlled by redox in bacteria is created by engineering a disulfide bond between the catalytic cysteine residue and the flanking polypeptide. A natural analog of this arrangement from Pyrococcus abyssi is found to respond to redox conditions in Escherichia coli, suggesting that inteins may function as biological sensors.

Hydrogen-deuterium exchange/mass spec analysis of PrP^Sc derived from mouse brains reveals that the infectious form of the prion adopts conformations that differ substantially from those previously postulated by various structural models.

A GFP-based reporter system has been developed to measure repair of DNA interstrand cross-links (ICLs) via homologous recombination. Cells defective in the Fanconi anemia pathway showed a marked defect in repair only when the reporter contained a replication origin, revealing an important role in ICL-induced homologous recombination coupled to replication.

Prior to induction, promoters tend to be depleted for nucleosomes, but the mechanism and function of this depletion has been obscure. It is now shown that in yeast, where a barrier can lead to phasing of nucleosomes, the degree of occupancy of these phased sites is predictably determined by the underlying DNA sequences; in addition, as occupancy is increased, nucleosomal removal upon induction is decreased, as is mRNA production.

The signal sequence on nascent peptides is recognized by the signal recognition particle (SRP), with subsequent targeting of the SRP-ribosomal nascent complex to the membrane. The structure of a signal sequence bound to the core SRP is presented, revealing structural changes in the SRP upon signal sequence binding.

Crystal structures of the protease domain of human HtrA1, with or without its PDZ domain, reveal that this enzyme's activity is controlled by substrate binding to its active site, via an induced-fit mechanism.