DNA binding and bending by the human papillomavirus type 16 E2 protein. Recognition of an extended binding site.
The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.
Thain
A
A
Department of Biochemistry, Centre for Molecular Recognition, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Webster
K
K
Emery
D
D
Clarke
A R
AR
Gaston
K
K
D001665:Binding Sites; D018834:Chaperonin 60 / Q000378:metabolism; D002942:Circular Dichroism; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D018834:Chaperonin 60; D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases