Mammalian transcription factor PBP. Characterization of its binding properties to the proximal sequence element of U6 genes.
The DNA binding properties of human transcription factor PBP, which specifically binds to the proximal sequence element of mammalian U6 genes and which plays a pivotal role during their transcription, were analyzed both qualitatively and quantitatively. As a prerequisite, we analyzed the optimal conditions for DNA binding of the PBP by assaying the stability of the interaction against increasing concentrations of salt, dithiothreitol, and heparin. The protein, which does not induce DNA bending, has a characteristic sensitivity against elevated temperatures and precipitously loses activity between 41 and 43 degrees C, a property which can be used for selective inactivation of the protein. Subjection of the PBP to limited proteinase K treatment showed that the protein consists of at least two functional domains, one of which is required for DNA binding. The PBP binds to the PSE with a much higher specific equilibrium constant (Ks = 1.33 x 10(11) M-1) than to nonspecific DNA (Kn = 1.18 x 10(5) M-1). The association and dissociation rates of PBP.PSE interactions were quantitatively determined by kinetic analyses. The pronounced lag phase during the initiation reaction of mammalian U6 transcription in vitro is probably correlated with the slow binding of the PBP to its target sequence. Once formed, however, the PBP.PSE complex is very stable and has a much lower dissociation (kd = 1.84 x 10(-5) s-1) than association rate constant (ka = 0.18 x 10(6) M-1 s-1). Collectively, the results demonstrate that the PSE binding protein stably associates with a high affinity to its cognate promoter sequence, and this process represents one of the primary events in the formation of the preinitiation complex on the U6 gene. Finally, we analyzed the effect of individual base pair mutations within mammalian U6 PSE sequences on the binding of the PBP.
Wanandi
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I
Institut für Molekularbiologie und Tumorforschung, Philipps Universität Marburg, Federal Republic of Germany.
Waldschmidt
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R
Seifart
K H
KH
D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D019286:Endopeptidase K; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D012342:RNA, Small Nuclear; D014157:Transcription Factors; D006493:Heparin; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D004229:Dithiothreitol