Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Chaperones assist the folding of many proteins in the cell. While the most well studied chaperones use cycles of ATP binding and hydrolysis to assist protein folding, a number of chaperones have been identified that promote protein folding in the absence of highenergy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we have characterized the kinetic mechanism of substrate folding by the small, ATP-independent chaperone, Spy. Spy rapidly associates with its substrate, Immunity protein 7 (Im7), eliminating its potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while remaining bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones can assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while continuously bound to a chaperone

Characterization of RNA aptamers that disrupt the RUNX1-CBFbeta/DNA complex

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor beta protein (CBFbeta). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD-CBFbeta complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity ( approximately 100-160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFbeta, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD-CBFbeta complex and thus will further aid exploring the functions of this key transcription factor