Association of human Pur alpha with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur alpha recognition element.
The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, a sequence-specific single-stranded DNA-binding protein implicated in control of gene transcription and DNA replication. These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Pur alpha or Rb. The Pur alpha-Rb complexes contain a form of Pur alpha with extensive post-synthetic modification, as demonstrated following expression of Pur alpha cDNA fused to a 9-amino acid epitope tag. Human Pur alpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Pur alpha binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Pur alpha recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Pur alpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats. Rb binding involves a Pur alpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur alpha to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.
Johnson
E M
EM
Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Chen
P L
PL
Krachmarov
C P
CP
Barr
S M
SM
Kanovsky
M
M
Ma
Z W
ZW
Lee
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005982:Glutathione Transferase; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000096:biosynthesis*; D017384:Sequence Deletion; D016324:Sequence Tagged Sites; D014157:Transcription Factors; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C501130:PURA protein, human; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D005982:Glutathione Transferase