Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA.
The E2 gene of bovine papillomavirus type 1 positively and negatively regulates the transcriptional enhancer located in the long control region of the viral genome. The DNA-binding domain of the E2 gene product was suspected to interact with the DNA sequence motif ACCN6GGT. We have shown that the carboxy-terminal 126 amino acids of the E2 protein constitute the DNA-binding domain. In this paper we described the expression of the E2 carboxy terminus in Escherichia coli and its subsequent purification. We provide definitive evidence that the protein recognizes the ACCN6GGT motifs in the viral enhancer. We show by methylation protection, methylation interference, and ethylation interference that the E2 protein contacts the DNA at the GG residues of the consensus sequence on both DNA strands. A gel retardation-DNase I footprint assay has revealed that the E2 DNA-binding domain exhibits different affinities for different ACCN6GGT motifs, indicating that nucleotides other than the conserved ACC and GGT sequences probably modulate the affinity of the DNA sequence for the E2 protein.
Moskaluk
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CA
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Bastia
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D
D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D008745:Methylation; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D014157:Transcription Factors