GATA1 directly mediates interactions with closely spaced pseudopalindromic but not distantly spaced double GATA sites on DNA.

The transcription factor GATA1 helps regulate the expression of thousands of genes involved in blood development, by binding to single or double GATA sites on DNA. An important part of gene activation is chromatin looping, the bringing together of DNA elements that lie up to many thousands of basepairs apart in the genome. It was recently suggested, based on studies of the closely related protein GATA3, that GATA-mediated looping may involve interactions of each of two zinc fingers (ZF) with distantly spaced DNA elements. Here we present a structure of the GATA1 ZF region bound to pseudopalindromic double GATA site DNA, which is structurally equivalent to a recently-solved GATA3-DNA complex. However, extensive analysis of GATA1-DNA binding indicates that although the N-terminal ZF (NF) can modulate GATA1-DNA binding, under physiological conditions the NF binds DNA so poorly that it cannot play a direct role in DNA-looping. Rather, the ability of the NF to stabilize transcriptional complexes through protein-protein interactions, and thereby recruit looping factors such as Ldb1, provides a more compelling model for GATA-mediated looping.

The cation-dependent G-proteins: in a class of their own.

G-proteins are some of the most important and abundant enzymes, yet their intrinsic nucleotide hydrolysis reaction is notoriously slow and must be accelerated in vivo. Recent experiments on dynamin and GTPases involved in ribosome assembly have demonstrated that their hydrolysis activities are stimulated by potassium ions. This article presents the hypothesis that cation-mediated activation of G-proteins is more common than currently realised, and that such GTPases represent a structurally and functionally unique class of G-proteins. Based on sequence analysis we provide a list of predicted cation-dependent GTPases, which encompasses almost all members of the TEES, Obg-HflX, YqeH-like and dynamin superfamilies. The results from this analysis effectively re-define the conditions under which many of these G-proteins should be studied in vitro.

Dihydroorotase from Escherichia coli: loop movement and cooperativity between subunits.

Escherichia coli dihydroorotase has been crystallized in the presence of the product, L-dihydroorotate (L-DHO), and the structure refined at 1.9A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105-115 has different conformations in the two subunits. In the case of the L-CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the L-DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient n=1.57(+/-0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.