A FLUORESCENCE ANISOTROPY STUDY OF DNA-BINDING BY HPV-11 E2C PROTEIN - A HIERARCHY OF E2-BINDING SITES

Association of the human papillomavirus (HPV) E2 protein with its pali ndromic DNA-binding site is a necessary step for transcriptional trans -activation. To study the interaction between DNA and E2, the carboxyl -terminal domain of HPV-11 E2 protein (E2C) was expressed in Escherich ia coli and purified to homogeneity, The binding affinity of the recom binant E2C protein for a single palindromic DNA recognition site was d etermined using a 5'-fluorescein-labeled 24 base pair oligonucleotide. Competitive titrations between the fluorescein-labeled oligonucleotid e and an unlabeled oligonucleotide of identical sequence yielded a nat ive affinity of 4.5 x 10(-9) M. Sequences from the seven E2-binding si r,, within the HPV-11 genome were titrated to establish a hierarchy of binding site affinities. All high-affinity E2-binding sites are locat ed within or near the HPV-11 LCR, E2-binding sites distant from the LC R appear to have low affinity for E2. When the location and affinity o f each E2-binding site an plotted in relation to a transcription map o f HPV-11, it is apparent that the major RNA transcripts produced refle ct the high-affinity E2-binding sites within the HPV LCR, To assess th e E2C-binding contribution of specific base pairs within the oligonucl eotide palindrome, additional double-stranded oligonucleotides were pr epared in which the central nonpalindromic sequences were varied. Whil e simple strand transposition of the A(4) . T-4 center had a minimal e ffect upon the E2C-oligonucleotide binding affinity, replacement with TATA . ATAT or CGCG . GCGC centers substantially decreased the affinit y of E2C for its binding site. Alteration of the canonical portions of the E2-binding palindrome reduced the DNA-protein binding affinity dr amatically.