Preferential interaction of the mRNA proofreading factor TFIIS zinc ribbon with rU·dA base pairs correlates with its function

The transcriptional factor TFIIS helps overcome elongation barriers and enhances proofreading by RNA polymerase II. These TFIIS functions may be modulated by the TFIIS zinc ribbon domain through interactions with nucleic acids in the elongation complex. Within this zinc ribbon domain, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have been shown to be critical for its function by mutant analysis. The sequence Asp261-Glu262 has been suggested to participate in metal binding within the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of electrostatic and stacking interactions. The interaction of the indole side chain of the tryptophan residue with nucleic acid bases is demonstrated by a characteristic and reversible decrease in the zinc ribbon fluorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM and no interaction at 500 mM NaCl), suggesting that the tryptophan stacking with nucleic acid base accompanies electrostatic contacts. The oligonucleotide−zinc ribbon interactions exhibit small but significant base preferences, as shown by the dependence of Keq on base composition, with decreasing Keq in the order U > T > A > C >> G. Within the variety of homopolymeric single- and double-stranded deoxy- and ribooligonucleotides, the oligonucleotide rU12-18·dA20 exhibited a 2−6-fold binding preference relative to other oligonucleotides. This preferential binding of the zinc ribbon to sequences composed of rU·dA base pairs, which are generally associated with elongation blocks, may help in overcoming elongation barriers. Since the mRNA proofreading and enhancement of elongation involve cleavage of ribonucleotide of the mismatched pair and the weakly paired rU·dA nucleotides, but not the stably paired rC·dG nucleotides, we propose that the Arg276-Trp277 sequence in the TFIIS zinc ribbon may serve as a scanner connected to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a criterion of determining their subsequent removal. The striking similarity in preference for mismatched and weakly paired nucleotides for binding and for excision suggests a functional relationship between binding and cleavage reactions

Stimulation of transcript elongation requires both the zinc finger and RNA polymerase II binding domains of human TFIIS

The eukaryotic transcriptional factor TFIIS enhances transcript elongation by RNA polymerase II. Here we describe two functional domains in the 280 amino acid human TFIIS protein: residues within positions 100-230 are required for binding to polymerase, and residues 230-280, which form a zinc finger, are required in conjunction with the polymerase binding region for transcriptional stimulation. Interestingly, a mutant TFIIS with only the polymerase binding domain actually inhibits transcription, whereas a mutant in which the polymerase binding and zinc finger domains are separated by an octapeptide is only weakly active. The zinc finger itself has no effect on transcription, but in contrast to the wild-type protein, it binds to oligonucleotides. These findings suggest that TFIIS may interact with RNA polymerase II such that the normally masked zinc finger can specifically contact nucleotides in the transcription elongation zone at a position juxtaposed to the polymerization site

Novel zinc finger motif in the basal transcriptional machinery : three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS

Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression. Here we describe homonuclear and lSN-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS. This domain contains a Cys4 ZnZ+- binding site with no homology to previously characterized Cys4, Cysa, or Cysz-His2 Zn fingers. Complete lH and I5N NMR resonance assignment of a 50-residue TFIIS peptideZnz+ complex is obtained. Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel p-sheet (designated the Zn ribbon). Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE. A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling. The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed