Stringent integrity requirements for both trans-activation and DNA-binding in a trans-activator, Oct3.
POU-specific and POU-homeo domains of Oct3 were produced in Echerichia coli for characterization of DNA binding to the octamer sequence. POU domain protein including A, B and H domains could bind to the octamer sequence efficiently and specifically, and DNase I footprint analysis gave an indistinguishable protection pattern between recombinant POU protein of Oct3 and native Oct3 from undifferentiated P19 cells. Truncated mutants, which contained B-specific and H domains or the H domain only, showed no binding activity, indicating that both of POU-specific and POU-homeo domains are essential for binding activity to octamer sequence. Furthermore, a 6 amino acid deletion from the N-terminal region of the A-specific domain is enough to destroy the binding activity. As for trans-activation, the N-terminal region is essential and sufficient. Deletion of the N-terminal proline-rich region rapidly eliminated trans-activating activity. These data strongly indicate the stringent integrity requirements for both trans-activation and DNA-binding domains in Oct3.
Imagawa
M
M
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Miyamoto
A
A
Shirakawa
M
M
Hamada
H
H
Muramatsu
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3; D010957:Plasmids / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA