Chromosomal translocations involving the N-terminal Ϸ250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.Ewings sarcoma oncogene ͉ EWS/ATF1 ͉ intrinsically disordered proteins ͉ EWS activation domain ͉ molecular recognition feature T he Ewings sarcoma (EWS) protooncogene together with TLS/FUS and hTAF II 68 form a subgroup (the TET family; ref. 1) within the RNP family of RNA-binding proteins. Aberrant chromosomal translocations involving EWS produce EWS fusion proteins (EFPs) that cause human cancers (2, 3) and EFPs share the following common features (Fig. 1A). First, all EFPs contain at least the N-terminal 7 exons of EWS (residues 1-264) encoding the EWS activation domain (EAD). Second, the EWS fusion partner is one of several cellular transcription factors containing a sequence-specific DNA-binding domain that specifies the tumor type. Third, EFPs are potent transcriptional activators that depend on both the EAD and the fusion partner. Gene-specific transcriptional deregulation is likely to be a primary route for tumorigenesis (2, 3). However, some biological effects of EFPs might occur by alternative mechanisms including perturbation of pre-mRNA splicing (3).Progress in determining structure/function relations for the EAD has been limited for several reasons. The EAD has a highly repetitive primary sequence (Fig. 1B) containing multiple copies of a degenerate hexap...