DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two 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 three proteins were specifically phosphorylated on Tyr701 in vitro and the phosphoprotein purified to homogeneity. This was achieved by employing a new isolation scheme that does not include DNA affinity steps and readily allows for the isolation of tens of milligrams of activated Stat protein. The purified phosphoprotein was free of traces of unphosphorylated polypeptide as detected by mass spectrometry. The phosphorylated Stat1 preparations bound to various DNA recognition sites with the same Keq of approximately 1 x 10(-9) M; distinction between 'weak' and 'strong' binding sites is determined by the very rapid dissociation (< 30 s, t1/2) from 'weak' sites compared with 'strong' sites (approximately 3 min, t1/2). Reports of 'weak' tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the finding that the Stat1 alpha or beta (38 amino acids shorter on the C terminus) 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 interaction.
Laboratory of Molecular Cell Biology, Rockefeller University, New York, NY 10021, USA.
U
U
Vinkemeier
S L
SL
Cohen
I
I
Moarefi
B T
BT
Chait
J
J
Kuriyan
J E
JE
Darnell
D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050832:Interferon-Stimulated Gene Factor 3; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C079868:gamma interferon activation factor; D004247:DNA; D066246:ErbB Receptors