No abstract

The ETS gene family encodes a group of more than 45 proteins, each with a highly conserved 85-amino acid DNA-binding domain initially mapped to v-ets-1, the member for which this group is named (1, 2). Members of the family identified to date share between 36 and 97% sequence identity with the Ets-1 DNA-binding domain and have been found in species ranging from lower invertebrates to humans. ETS family members can function as transcriptional activators or repressors and are involved in a wide range of tissue specific developmental processes. In humans, they are involved in hematopoiesis (3-5), ossification (6, 7), myogenisis (8), and angiogenesis (9, 10). ETS proteins have also been implicated in several types of cancer and other human diseases (11). Because ETS proteins have overlapping DNA binding specificities and because their expression is often tissue type-specific, inappropriate expression or altered forms of a given ETS protein will likely activate genes that are normally not expressed. Thus, an understanding of the sequence specificity of ETS proteins is important for understanding the mechanism of deregulation in ETS-related cancers.All ETS DNA-binding domains recognize a purine-rich G-G-A sequence, yet each family member has specificity for characteristic bases flanking this segment (2,(12)(13)(14)(15)(16)(17)(18)(19). The recent solution and crystal structures of the ETS domains of Fli-1 (20), Ets-1 (21-23), PU.1 (24), GABP␣/␤ (25), SAP-1 (26), and Elk-1 (27) have established that the ETS proteins constitute a subgroup within the super-family of "winged helix-loop-helix" DNA-binding proteins. Structures of these six domains complexed with DNA show that helix-3 lies in the major groove centered at the G-G-A recognition site. In the crystal structures of PU.1 (24), GABP␣ (25), SAP-1 (26), and Elk-1 (27), two conserved Arg residues within helix 3 make direct hydrogen bonds with the bases of the G-G-A motif. Importantly, the pattern of hydrogen bonds from these conserved arginines is not the same in the different high resolution crystal structures. This suggests that the ETS domain may have some degree of flexibility and diversity in its mode of interaction with DNA. Regions of the ETS domain flanking helix-3 interact with phosphates along the minor groove both upstream and downstream of the G-G-A element, further stabilizing the complex and bending the DNA around the protein. Variation in DNA bending from 11 to 28°for the SAP-1 and PU.1 complexes, respectively, and few unique direct contacts to the bases flanking G-G-A suggest a possible "indirect read-out" mechanism of DNA recognition, wherein the ETS domain recognizes a sequence-dependent structure that is either induced or already present in DNA. This model is in contrast to a "direct read-out" mechanism of DNA binding specificity, in which protein residues recognize and interact with unique base pairs within an ETS-binding site. In both mechanisms water molecules could potentially mediate contacts between the protein and the DNA.To better understand...

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13 C, 15 N enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH 4 + , glucose and CO 2 as stoichiometric precursors in labeled form. Using this approach, U-13 C-GTP, U-13 C, 15 N-GTP, 13 C 2,8 -ATP and U-15 N-GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region (TAR) RNA containing 13 C 2,8 -adenosine and 15 N-1,3,7,9,2 -guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected expanding the applicability of enzymatic synthesis.