A fluorescence anisotrophy study of DNA binding by HPV-11 E2C protein: A hierarchy of E2-binding sites

被引:31
作者
Alexander, KA [1 ]
Phelps, WC [1 ]
机构
[1] GLAXO WELLCOME CO, DEPT MOL & CELLULAR VIROL, RES TRIANGLE PK, NC 27709 USA
关键词
D O I
10.1021/bi960447d
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Association of the human papillomavirus (HPV) E2 protein with its palindromic 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 Escherichia coli and purified to homogeneity, The binding affinity of the recombinant E2C protein for a single palindromic DNA recognition site was determined using a 5'-fluorescein-labeled 24 base pair oligonucleotide. Competitive titrations between the fluorescein-labeled oligonucleotide and an unlabeled oligonucleotide of identical sequence yielded a native affinity of 4.5 x 10(-9) M. Sequences from the seven E2-binding sir,, within the HPV-11 genome were titrated to establish a hierarchy of binding site affinities. All high-affinity E2-binding sites are located within or near the HPV-11 LCR, E2-binding sites distant from the LCR appear to have low affinity for E2. When the location and affinity of each E2-binding site an plotted in relation to a transcription map of HPV-11, it is apparent that the major RNA transcripts produced reflect the high-affinity E2-binding sites within the HPV LCR, To assess the E2C-binding contribution of specific base pairs within the oligonucleotide palindrome, additional double-stranded oligonucleotides were prepared in which the central nonpalindromic sequences were varied. While simple strand transposition of the A(4) . T-4 center had a minimal effect upon the E2C-oligonucleotide binding affinity, replacement with TATA . ATAT or CGCG . GCGC centers substantially decreased the affinity of E2C for its binding site. Alteration of the canonical portions of the E2-binding palindrome reduced the DNA-protein binding affinity dramatically.
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页码:9864 / 9872
页数:9
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