High affinity interaction of yeast transcriptional regulator, Mot1, with TATA box-binding protein (TBP).
Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202, USA.
J I
JI
Adamkewicz
K E
KE
Hansen
W A
WA
Prud'homme
J L
JL
Davis
J
J
Thorner
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D000251:Adenosine Triphosphatases; C073676:MOT1 protein, S cerevisiae; D004265:DNA Helicases