Pax6, a transcription factor containing the bipartite paired DNA-binding domain, has critical roles in development of the eye, nose, pancreas, and central nervous system. The 2.5 Å structure of the human Pax6 paired domain with its optimal 26-bp site reveals extensive DNA contacts from the amino-terminal subdomain, the linker region, and the carboxy-terminal subdomain. The Pax6 structure not only confirms the docking arrangement of the amino-terminal subdomain as seen in cocrystals of the Drosophila Prd Pax protein, but also reveals some interesting differences in this region and helps explain the sequence specificity of paired domain-DNA recognition. In addition, this structure gives the first detailed information about how the paired linker region and carboxy-terminal subdomain contact DNA. The extended linker makes minor groove contacts over an 8-bp region, and the carboxy-terminal helix-turn-helix unit makes base contacts in the major groove. The structure and docking arrangement of the carboxy-terminal subdomain of Pax6 is remarkably similar to that of the amino-terminal subdomain, and there is an approximate twofold symmetry axis relating the polypeptide backbones of these two helix-turn-helix units. Our structure of the Pax6 paired domain-DNA complex provides a framework for understanding paired domain-DNA interactions, for analyzing mutations that map in the linker and carboxy-terminal regions of the paired domain, and for modeling protein-protein interactions of the Pax family proteins.

The highly conserved Paf1 complex (PAF1C) plays critical roles in RNA polymerase II transcription elongation and in the regulation of histone modifications. It has also been implicated in other diverse cellular activities, including posttranscriptional events, embryonic development and cell survival and maintenance of embryonic stem cell identity. Here, we report the structure of the human Paf1/Leo1 subcomplex within PAF1C. The overall structure reveals that the Paf1 and Leo1 subunits form a tightly associated heterodimer through antiparallel beta-sheet interactions. Detailed biochemical experiments indicate that Leo1 binds to PAF1C through Paf1 and that the Ctr9 subunit is the key scaffold protein in assembling PAF1C. Furthermore, we show that the Paf1/Leo1 heterodimer is necessary for its binding to histone H3, the histone octamer, and nucleosome in vitro. Our results shed light on the PAF1C assembly process and substrate recognition during various PAF1C-coordinated histone modifications.

Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. Here we discover that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.

The evolutionarily conserved Elongator complex, which is composed of six subunits elongator protein 1 (Elp1 to -6), plays vital roles in gene regulation. The molecular hallmark of familial dysautonomia (FD) is the splicing mutation of Elp1 [also known as IκB kinase complex-associated protein (IKAP)] in the nervous system that is believed to be the primary cause of the devastating symptoms of this disease. Here, we demonstrate that disease-related mutations in Elp1 affect Elongator assembly, and we have determined the structure of the C-terminal portion of human Elp1 (Elp1-CT), which is sufficient for full-length Elp1 dimerization, as well as the structure of the cognate dimerization domain of yeast Elp1 (yElp1-DD). Our study reveals that the formation of the Elp1 dimer contributes to its stability in vitro and in vivo and is required for the assembly of both the human and yeast Elongator complexes. Functional studies suggest that Elp1 dimerization is essential for yeast viability. Collectively, our results identify the evolutionarily conserved dimerization domain of Elp1 and suggest that the pathological mechanisms underlying the onset and progression of Elp1 mutation-related disease may result from impaired Elongator activities.F amilial dysautonomia (FD) (Online Mendelian Inheritance in Man: 223900), which is an Ashkenazi Jewish disorder with an incidence of 1:3,700 live births, is an autosomal recessive disorder that affects the development and survival of sensory, sympathetic, and some parasympathetic neurons (1). FD patients are characterized by cardiovascular instability, gastrointestinal dysfunction, vomiting crises, decreased sensitivity to pain and temperature, and recurrent pneumonias (2). The most common mutation, which represents >99.5% of all FD patients, is a T-to-C transition in position six of the donor splice site of intron 20 in the gene transcript of the IκB kinase (IKK) complex-associated protein (IKAP), also known as elongator protein 1 (Elp1) (3, 4). This splicing mutation causes skipping of exon 20 and results in encoding a truncated IKAP/Elp1 protein (referred to as Elp1-FD hereafter) (Fig. 1A).IKAP/Elp1 was originally identified as a scaffold protein and a regulator for IKKs involved in proinflammatory cytokine signaling (5). However, contrary to this observation, subsequent analyses failed to demonstrate an association with IKKs (6). Coincident and subsequent studies supported the notion that IKAP/Elp1 is a component of the highly conserved Elongator complex from different species (7-9) (for simplicity, the term Elp1 will be used in the following discussion). The holo-Elongator complex contains six subunits (Elp1 to -6) and assembles into a core subcomplex (Elp1 to -3) and an accessory subcomplex (Elp4 to -6), which is involved in substrate recognition (10, 11). Moreover, the holo-Elongator is a functional unit, as illustrated in yeast, in which strains lacking any of the six Elp proteins exhibit similar phenotypes (7,12,13), and in that the removal of any Elongator subunits affects the...

Inspired by the inertial proximal algorithms for finding a zero of a maximal monotone operator, in this paper, we propose two inertial accelerated algorithms to solve the split feasibility problem. One is an inertial relaxed-CQ algorithm constructed by applying inertial technique to a relaxed-CQ algorithm, the other is a modified inertial relaxed-CQ algorithm which combines the KM method with the inertial relaxed-CQ algorithm. We prove their asymptotical convergence under some suitable conditions. Numerical results are reported to show the effectiveness of the proposed algorithms.