DNA-binding studies of XSPTSPSZ, derivatives of the intercalating heptad repeat of RNA polymerase II

Abstract

The synthesis, solution conformation, and interaction with DNA of three 8-residue peptides structurally related to the heptad repeat unit found at the C-terminus of RNA polymerase II are reported. Peptides QQ, XQ, and PQ are derived from the parent sequence YSPTSPSY (peptide YY), which was reported to bind to DNA by bisintercalation [M. Suzuki (1990) Nature, Vol. 344, pp. 562- 565], and contain either a 2-quinolyl (Q), 2-quinoxolyl (X), or 5- phenanthrolyl (P) group in place of the aromatic side chains of the N-and C- terminal tyrosine residues present in the parent sequence. The combined results of linear dichroism and induced CD measurements of peptides QQ, XQ, and PQ with calf thymus DNA are consistent with weak binding of the peptides to DNA in a preferred orientation in which the chromophores are intercalated. Small increases in the melting temperatures of poly [d(A-T)2] are also consistent with the peptides interacting with DNA. While enzymatic footprinting with DNase I showed no protection from cleavage by the enzyme, chemical footprinting with fotemustine showed that the peptides modify the reactivity of the major groove, presumably via minor groove binding. Peptide QQ inhibited fotemustine alkylation significantly more than either XQ or PQ, and slightly more than YY. In aqueous solution, nmr experiments on QQ, XQ, and PQ show a significant population of a conformation in which Ser2-Pro3- Thr4-Ser5 form both type I and type H β-turn conformations in equilibrium with open chain conformations. Nuclear magnetic resonance titration experiments of PQ with (GCGTACGC)2 showed small changes in chemical shifts, consistent with the formation of a weak nonspecific complex. Analogous experiments, using peptides QQ and XQ with (GCGTACGC)2, and peptide YY with (CGTACG)2, showed no evidence for the interaction of the peptides with these oligonucleotides. These results show that peptides of general structure XSPTSPSZ are weak nonspecific DNA binders that differ significantly from previously characterized S(T)PXX DNA-binding motifs that are generally AT- selective minor groove binders. Show more