Abstract
The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.
Highlights
The ETS transcription factors play roles in normal cellular processes as well as a variety of human malignancies
The double mutant (A94D/V113E), termed TELM, appeared to be monomeric based on its chromatographic properties. These findings suggested that wild-type, full-length TEL has self-association properties similar to the well characterized PNT domain-only fragment and that the monomeric TELM species was suitable for further analysis
We have demonstrated that TEL DNA binding is repressed by autoinhibition
Summary
Expression Plasmids—Murine TEL cDNA was cloned into the bacterial expression vector pET28B (Novagen) and used for generation of mutant versions (A94D, V113E, and A94D/ V113E) by site-directed mutagenesis. Complimentary oligonucleotides were designed to contain a single ETS binding site (underlined) and no additional GGA sequences: 5Ј-CACGCCGCATGTATGTAGCCTGTTGCTAGCTGCCGGAAGTAATACGTAACGCAGCTATTGCTACTAACTATTGTGCTGCA-3Ј (top strand). The DNA duplexes were digested with PstI (5Ј) and KpnI (3Ј) and inserted into the pKS-EBS1 to generate a series of pKS-EBS2 vectors with appropriately spaced binding sites. Equilibrium binding conditions were set up with a series of protein dilutions (10Ϫ13 to 10Ϫ7 M) mixed with radiolabeled DNA duplexes (2.5 ϫ 10Ϫ12 M) and incubated at room temperature for 1 h to reach equilibrium. Binding reactions consisting of protein (10Ϫ8 M) and radiolabeled DNA duplex (10Ϫ10 M) were incubated at room temperature for ϳ1 h. To the remaining reaction mixture, a 1000-fold excess unlabeled 27-bp DNA duplex containing a single ETS binding site in 1/10 volume of EMSA buffer was added. For analysis by electrospray ionization/mass spectrometry (ESI/MS), TEL species ⌬N334:⌬C436, and ⌬N334: ⌬C436 (E431A and E434A) were digested with 50 ng of trypsin under conditions described above, and the reactions were stopped by the addition of 1% trifluoroacetic acid
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
