PREFERENTIAL INTERACTION OF THE MESSENGER-RNA PROOFREADING FACTOR TFIIS ZINC RIBBON WITH RU-CENTER-DOT-DA BASE-PAIRS CORRELATES WITH ITS FUNCTION

The transcriptional factor TFIIS helps overcome elongation barriers an d enhances proofreading by RNA polymerase II. These TFIIS functions ma y be modulated by the TFIIS zinc ribbon domain through interactions wi th nucleic acids in the elongation complex. Within this zinc ribbon do main, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have bee n shown to be critical for its function by mutant analysis. The sequen ce Asp261-Glu262 has been suggested to participate in metal binding wi thin the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of el ectrostatic and stacking interactions. The interaction of the indole s ide chain of the tryptophan residue with nucleic acid bases is demonst rated by a characteristic and reversible decrease in the zinc ribbon f luorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM a nd no interaction at 500 mM NaCl), suggesting that the tryptophan stac king with nucleic acid base accompanies electrostatic contacts. The ol igonucleotide-zinc ribbon interactions exhibit small but significant b ase preferences, as shown by the dependence of K-eq on base compositio n, with decreasing K-eq in the order U > T > A > C much greater than G . Within the variety of homopolymeric single- and double-stranded deox y- and ribooligonucleotides, the oligonucleotide rU(12-18).dA(20) exhi bited a 2-6-fold binding preference relative to other oligonucleotides . This preferential binding of the zinc ribbon to sequences composed o f rU.dA base pairs, which are generally associated with elongation blo cks, may help in overcoming elongation barriers. Since the mRNA proofr eading and enhancement of elongation involve cleavage of ribonucleotid e of the mismatched pair and the weakly paired rU.dA nucleotides, but not the stably paired rC.dG nucleotides, we propose that the Arg276-Tr p277 sequence in the TFIIS zinc ribbon may serve as a scanner connecte d to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a cri terion of determining their subsequent removal. The striking similarit y in preference for mismatched and weakly paired nucleotides for bindi ng and for excision suggests a functional relationship between binding and cleavage reactions.