Research articles

Studying protein interactions at membranes is a technical challenge. A quantitative approach to measuring the interaction between the apoptotic proteins tBid and Bcl using fluorescence correlation spectroscopy reveals that membranes have an active role in modulating BCL2 protein interactions.

Despite the importance of small RNA–mediated silencing, no structural information exists for complexes of known function. Using single-particle EM, the structure of the minimal functional unit for RNAi in humans (AGO2, Dicer and TRBP) is now presented.

Increasing evidence indicates that membrane protein function can be affected by the surrounding membrane bilayer. A new study on voltage-gated potassium channels using tarantula toxins suggests that lipid interaction with the voltage sensor can influence channel function.

F₁F_o ATP synthases produce ATP using proton- or sodium-motive force to drive ions through the membrane-embedded F_o complex, causing rotation of its c-ring rotor leading to ATP synthesis. The first high-resolution crystal structure of the c-ring from a proton-translocating F₁F_o-ATP synthase reveals the architecture of the proton-binding site and provides insight into the mechanism of proton transport.

Some p53 mutations result in gain-of-function variants that can contribute to tumorigenesis. Three such mutants, R175H, R273H and R280K p53, are now shown to cooperate with transcription factor E2F1 to upregulate the expression of ID4, which in turn stabilizes the transcripts from pro-angiogenic factors IL-8 and GRO-α.

Identifying physiological substrates of proteases still poses a challenge. An unbiased approach using the heterologous Escherichia coli proteome now identifies the structural and sequence determinants for caspase-3 substrates, revealing a kinetic threshold that can distinguish relevant substrates.

Transcription initiation involves recruitment of key factors to promoters. Yeast TATA-binding protein (TBP) turnover is now examined genome-wide and genes transcribed by the three RNA polymerases found to have distinct signatures. Further analyses suggest that TBP dynamics, rather than DNA sequence affinity per se, is key to gene expression.

Rhesus macaque monkeys can inhibit retroviral replication via TRIMCyp, a variant of TRIM5a with an insertion of the cyclophilin A cDNA. Cyclophilin A binds to HIV-1 capsid, whereas TRIMCyp restricts HIV-2. How the change in specificity of this domain occurred is now revealed through biophysical and structural studies.

An improved method for detecting proteins phosphorylated by the ERK kinase reveals multiple new in vitro ERK substrates, including three nucleoporin proteins. Nup50 is phosphorylated in FG repeats by ERK2 in vivo and in vitro, suggesting a new mechanism by which MAP kinase signaling controls nuclear translocation of proteins.

Colicins are secreted bacterial toxins. To avoid killing the producer organism, each colicin is coexpressed with a high-affinity inhibitor, or immunity protein (Im). The evolution of Im-Colicin interfaces and the evolvability traits of protein-protein interactions are now examined using in vitro evolution and structural analyses.

The maturation of tRNAs involves folding into their L shape and nucleotide modifications at several positions. Some modifying enzymes require an L-shaped substrate, and the crystal structure of methylase Trm5 in complex with AdoMet and tRNA now reveals how the substrate tertiary structure is sensed.

Alternative splicing increases genome coding potential and is affected by factors including the hnRNPs. The effect of altering splice site strength on splicing activity is now found to be antagonized by nearby hnRNP H binding sites. Other splicing factor sites may have similar effects and may thus have influenced splice form evolution.

Escherichia coli YiiP, a member of the cation diffusion facilitator family, exports cytoplasmic zinc, maintaining cellular homeostasis. The high-resolution crystal structure of YiiP, combined with functional studies focused on its cytoplasmic C-terminal domain, suggest how it is able to allosterically modulate zinc transport.

The positions of nucleosomes can affect processes occurring on DNA. DNA curtains are now used to study nucleosome positioning in vitro. This allows assessment of sequence-related effects on positioning and indicates that the yeast factor Scm3 can overcome the aversion of nucleosomes to AT-rich sequences.

Nucleosomes can be modified by replacing the core histones with variants, the most diverse of which is macroH2A. The localization of macroH2A variants in human male pluripotent cells indicates that this variant functions in repression of key developmental genes and is essential for zebrafish embryogenesis.

DNA polymerase δ (Pol δ) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol δ catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-Å resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

The let-7 microRNA has been implicated in development and disease. Its expression must thus be tightly regulated, and previously uridylation and Lin28 were implicated in let-7 stability. Zcchc11 is now shown to be the uridylase that mediates pre–let-7 modification and regulates mature let-7 levels and activity in mouse embryonic stem cells.

Developmental expression of the microRNA let-7 is tightly regulated in many animals, and turnover has been linked to LIN-28 and uridylation in mammals. This regulation is now shown to be conserved in Caenorhabditis elegans, and PUP-2 is shown to be a uridylase that is specifically recruited to let-7 in a LIN-28–dependent manner.

Nucleosomes can interfere with DNA binding by factors, but previous work showed that protein-binding sites on a single nucleosome are accessible. Dynamics in the context of higher-order chromatin structure are now examined, with compaction dynamics and DNA-binding site exposure on a centrally placed nucleosome in an array assessed.

Co-transcriptional splicing of pre-mRNAs has been proposed to involve exon tethering to the elongating RNA polymerase II. By inserting a fast-cleaving ribozyme in the nascent transcript, the linear integrity of the transcript is found to be key to splicing, arguing against tethering and for a pathway that clears such disrupted transcripts.