DNA-BINDING OF IN-VITRO ACTIVATED STAT1-ALPHA, STAT1-BETA AND TRUNCATED STAT1 - INTERACTION BETWEEN NH2-TERMINAL DOMAINS STABILIZES BINDINGOF 2 DIMERS TO TANDEM DNA SITES

Stat1 alpha, Stat1 beta and a proteolytically defined truncated Stat1 (132-713, Stat1tc) have been prepared from recombinant sources, All th ree proteins were specifically phosphorylated on Tyr701 in vitro and t he phosphoprotein purified to homogeneity, This was achieved by employ ing a new isolation scheme that does not include DNA affinity steps an d readily allows for the isolation of tens of milligrams of activated Stat protein, The purified phosphoprotein was free of traces of unphos phorylated polypeptide as detected by mass spectrometry, The phosphory lated Stat1 preparations bound to various DNA recognition sites with t he same K-eq of similar to 1x10(-9) M; distinction between 'weak' and 'strong' binding sites is determined by the very rapid dissociation (< 30 s, t(1/2)) from 'weak' sites compared with 'strong' sites (similar to 3 min, t(1/2)), Reports of 'weak' tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the find ing that the Stat1 alpha or beta (38 amino acids shorter on the C term inus) bound to two tandem sites (but not two head-to-head sites) with a higher stability than to a single recognition site, The N-terminally truncated protein Stat1tc did not show this cooperative binding, thus implicating the N-terminal domain in promoting Stat1-Stat1 dimer inte raction.