Research articles

Amyloid fibrils feature in many human diseases and in epigenetic memory, but understanding their molecular structure has been difficult. Now, through a combination of optical trapping and fluorescent imaging to examine amyloid fibrils of the yeast prion protein Sup35, the unexpected unfolding of individual subdomains has been detected, suggesting strong noncovalent interactions maintain the fibril even if individual monomers unfold.

Resection of DNA double strands is known to require a number of factors, but the exact roles each one plays in the process are still unclear. Now the resection events in yeast are reconstituted biochemically, showing that the MRX complex and Sae2 directly stimulate the resection of 5' strands by Exo1.

Dom34 and Hbs1 are involved in no-go decay (NGD) and nonfunctional 18S rRNA decay (18S NRD) pathways that eliminate RNAs causing translation stalling. Now structural work reveals the similarity of the Dom34–Hbs1 complex with elongation factor–tRNA and translation termination eRF1–eRF3 complexes. Mutagenesis analysis of Hbs1 shows that NGD and 18S NRD can be genetically uncoupled.

Reverse transcriptase is an essential enzyme for HIV replication. Single-molecule studies reveal for the first time the structural dynamics of the reverse transcription initiation complex in real time. Reverse transcriptase can bind its tRNA–vRNA substrate in two opposite orientations and flip between these two states.

Alterations in BRCA1 transcription can contribute to sporadic forms of breast cancer. Now the dynamics between transcriptional coactivators and co-repressors at the BRCA1 promoter reveal a central role for the metabolic sensor CtBP in the response to estrogen or cellular metabolic status

Despite recognizing an epitope on HIV-1 gp41 that partially overlaps with those from broadly neutralizing antibodies, mAb 13H11 is non-neutralizing. Now the crystal structure and binding studies of 13H11 Fab with a gp41 peptide reveal why: the antigen assumes a helical structure consistent with the post-fusion conformation of gp41.

A number of antibodies against the membrane-proximal region of HIV-1 gp41 cannot neutralize the virus, despite their high affinity. Now binding studies along with a crystal structure indicate that these non-neutralizing antibodies recognize gp41 in a post-fusion conformation.

A high-resolution structure of an off-pathway misfolded intermediate state of a PDZ domain is now obtained, through a combination of phi-value analysis and computational modeling. The structure reveals that a misfolded intermediate can look quite similar to the native state.

Mutations in BRCA2 are associated with higher susceptibility to some forms of cancer. BRCA2 is known to play a central role in the repair of DNA breaks via homologous recombination. Now a role for BRCA2 in telomere integrity is revealed, indicating that BRCA2 can contribute to genome stability in multiple ways.

Chromatin carries various modifications that have been related to DNA and RNA metabolism. Analysis of numerous histone modifications across the human genome now demonstrates that intronic and exonic regions are enriched with distinct modifications. Studies of two alternatively spliced genes suggest that exon definition, rather than splicing, may contribute to these patterns.

Nucleosomes consist of two copies of each histone. H2A.Z is a variant H2A-related histone known to be enriched around transcription start sites. However, an H2A.Z-containing nucleosome could contain contain two copies of H2A.Z (homotypic) or one of H2A.Z and one of canonical H2A (heterotypic). Homotypic and heterotypic H2A.Z nucleosomes are now mapped and their distributions relative to promoters analyzed.

tRNA and mRNA translocation on the ribosome is catalyzed by the GTPase EF-G. Using single-molecule FRET analysis, this process is examined, indicating that GTP hydrolysis does not affect translation through promoting a ribosomal unlocked state, but that constraining the conformation of EF-G to a high-affinity interaction with the ribosome is key to translocation rate.

In yeast cells, short telomeres are preferentially elongated by the telomerase complex in a process that requires Tel1. Now low levels of telomerase inhibitory protein Rif2 and the absence of Mec1 are shown to mark short telomeres for Tel1 binding and elongation rather than double-strand break repair.

The anthrax lethal toxin protein transporter consists of protective antigen (PA) and lethal factor (LF), with LF unfolding for translocation into the host cell. Structural and functional analyses now indicate how each of four LFs unfolds and binds into amphipathic clefts on the surface of the PA octomer.

Open channel blockers are very different in their chemistry and structure. Now GLIC, a bacterial model for pentameric ligand-gated channels, is used to investigate the structural basis of open channel block. The work identifies two extended interaction sites, with large blockers binding to the center of the membrane and small blockers binding to the narrow intracellular region.

A genome-wide analysis of replication profiles in different budding yeast mutants reveals that the alternative clamp loader component Ctf18 has a key role in the DNA replication checkpoint, acting together with Mrc1 to promote Rad53 phosphorylation.

Notch signaling regulates key developmental events, and aberrant Notch signaling has been linked to disease. The structure of a dimeric Notch transcription complex with DNA is presented, revealing how dimer-binding sites with different spacers may be recognized. Cryptic dimer-binding sites are also uncovered, which might account for differential sensitivity of promoters to cooperative assembly of complexes.

Vesicle tethering is the least well understood step of vesicular transport. The core conserved oligomeric Golgi (COG) vesicle tethering complex from yeast, consisting of the four essential subunits Cog1–4, is now reconstituted and characterized by negative stain EM and single particle analysis. The COG complex forms a y-shaped structure, whose central region and the distal regions of at least two legs all participate in interactions with other components of the intracellular trafficking machinery.

How a variety of nuclear export signals (NESs) are recognized by their receptor CRM1 is now examined by crystallography, NMR spectroscopy, biochemistry and cellular approaches. The work reveals that the different NES peptides adopt different conformations to fit into five rigid hydrophobic binding pockets on CRM1, and redefines an NES consensus.

Alternative splicing can be affected by transcription elongation rate and genotoxic stress. Camptothecin is now shown to induce MDM2 exon skipping in a p53-independent and reversible manner. This process involves disrupting EWS and YB-1 interaction, which are Pol II and spliceosome-associated factors, respectively.